Novel influenza hemagglutinin protein-based vaccines

ABSTRACT

Novel vaccines are provided that elicit broadly neutralizing anti-influenza antibodies. Some vaccines comprise nanoparticles that display hemagglutinin trimers from influenza virus on their surface. The nanoparticles comprise fusion proteins comprising a monomeric subunit of ferritin joined to at least a portion of an influenza hemagglutinin protein. Some portions comprise the ectodomain while some portions are limited to the stem region. The fusion proteins self-assemble to form the hemagglutinin-displaying nanoparticles. Some vaccines comprise only the stem region of an influenza hemagglutinin protein joined to a trimerization domain. Such vaccines can be used to vaccinate an individual against infection by heterologous influenza viruses and influenza virus that are antigenically divergent from the virus from which the nanoparticle hemagglutinin protein was obtained. Also provided are fusion proteins and nucleic acid molecules encoding such proteins. Finally, also provided are assays using nanoparticles of the invention to detect anti-influenza antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to P.C.T. Patent Application Serial No. PCT/US12/56822 entitled “NOVEL INFLUENZA HEMAGGLUTININ PROTEIN-BASED VACCINES” filed Sep. 24, 2012; which claims priority to U.S. Provisional Patent Application Ser. No. 61/661,209 entitled “SELF-ASSEMBLED FERRITIN NANOPARTICLES EXPRESSING HEMAGGLUTININ AS AN INFLUENZA VACCINE” filed Jun. 18, 2012; and U.S. Provisional Patent Application Ser. No. 61/538,663 entitled “SELF-ASSEMBLED FERRITIN NANOPARTICLES EXPRESSING HEMAGGLUTININ AS AN INFLUENZA VACCINE” filed Sep. 23, 2011, all of which are incorporated herein in their entirety by this reference.

SUMMARY OF THE INVENTION

The present invention provides novel hemagglutinin protein-based influenza vaccines that are easily manufactured, potent, and which elicit broadly neutralizing influenza antibodies. In particular, the present invention provides influenza hemagglutinin proteins, and portions thereof, that are useful in inducing the production of neutralizing antibodies. It also provides novel HA-ferritin nanoparticle (np) vaccines. Such nanoparticles comprise fusion proteins, each of which comprises a monomeric subunit of ferritin joined to an immunogenic portion of an influenza hemagglutinin protein. Because such nanoparticles display influenza hemagglutinin protein on their surface, they can be used to vaccinate an individual against influenza virus.

In one embodiment, the invention is a nanoparticle that comprises a fusion protein, and in this embodiment the fusion protein comprises at least 25 contiguous amino acids from a monomeric ferritin subunit protein joined to a first influenza hemagglutinin (HA) protein, such that the nanoparticle comprises influenza virus HA protein trimers on its surface. The nanoparticle can form an octahedron, which can consist of 24 subunits having 432 symmetry. Further, the monomeric ferritin subunit protein can be selected from a bacterial ferritin, a plant ferritin, an algal ferritin, an insect ferritin, a fungal ferritin and a mammalian ferritin, and in a preferred embodiment, is a Helicobacter pylori ferritin protein.

In this embodiment, the monomeric ferritin subunit protein can comprise at least 25 contiguous amino acids of an amino acid sequence selected from SEQ ID NO:2 and SEQ ID NO:5 or can comprise an amino acid at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 97% identical, at least about 99% identical to those sequences or can comprise those sequences. In another embodiment, the monomeric subunit comprises a region corresponding to amino acids 5-167 of SEQ ID NO:2.

In this embodiment, the hemagglutinin protein can comprise at least 25 contiguous amino acids from the hemagglutinin protein of an influenza virus selected from A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009 CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968 (1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo, B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris, H1), B/Brisbane/60/2008 (2008 Bris, B). Also, the hemagglutinin protein can comprise an amino acid sequence that is selected from the amino acid sequences of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38 or one that is at least 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 97% identical, at least about 99% identical thereto. Alternatively, the hemagglutinin protein can comprise an amino acid sequence that is selected from the amino acid sequences of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98 or one that is at least 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 97% identical, at least about 99% identical thereto.

In this embodiment, the hemagglutinin protein can be capable of eliciting an immune response to a protein comprising an amino acid sequence selected from SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38 or it can comprise a region selected from a region capable of allowing formation of a hemagglutinin trimer, a stem region, an ectodomain, and a region comprising the amino acid sequence from the amino acid residue immediately distal to the last amino acid of the second helical coiled coil to the amino acid residue proximal to the first amino acid of the transmembrane domain.

The hemagglutinin protein can also comprise a hemagglutinin spike domain, a region corresponding to amino acids 1-519 of SEQ ID NO:8 or an amino acid sequence selected from the group consisting of amino acids 1-519 of SEQ ID NO:8 and SEQ ID NO:11.

In this embodiment, the fusion protein can comprise a linker sequence.

In this embodiment, the nanoparticle can elicit an immune response against a stem region of influenza hemagglutinin, a spike of influenza hemagglutinin, an influenza virus strain that is heterologous to the strain influenza virus from which the hemagglutinin protein was obtained or an influenza virus that is antigenically divergent from the influenza virus from which the hemagglutinin protein was obtained.

In this embodiment, the fusion protein can comprise an amino acid sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 97% identical, at least about 99% identical to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68, wherein the nanoparticle elicits an immune response against an influenza virus or can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68. The fusion protein can also comprise an amino acid sequence at least 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 97% identical, at least about 99% identical to a sequence selected from the group consisting of SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128, wherein the nanoparticle elicits an immune response against an influenza virus.

In this embodiment, the nanoparticle can comprise a second fusion protein comprising a second influenza hemagglutinin protein, wherein the first and second influenza hemagglutinin proteins are from different Types, from different sub-types or different strains of influenza viruses.

Another embodiment of the present invention is a vaccine composition comprising any of the foregoing nanoparticle. The vaccine composition can further comprise at least one additional nanoparticle that comprises at least one hemagglutinin protein from a different strain of influenza than the first hemagglutinin protein and the second hemagglutinin protein.

A further embodiment of the invention is a method to produce a vaccine against influenza virus. The method includes expressing a fusion protein comprising a monomeric ferritin protein joined to an influenza hemagglutinin protein under conditions such that the fusion proteins form a nanoparticle displaying hemagglutinin trimers on its surface and recovering the nanoparticle.

The invention also includes a method to vaccinate an individual against influenza that includes administering a nanoparticle to an individual such that the nanoparticle elicits an immune response against influenza virus. In this embodiment, the nanoparticle comprises a monomeric subunit of ferritin joined to an influenza hemagglutinin protein and the nanoparticle displays influenza hemagglutinin trimers on its surface. In this embodiment, the nanoparticle can elicit an immune response to an influenza virus strain that is heterologous to the sub-type or strain of or that is antigenically divergent from the influenza virus from which the hemagglutinin protein was obtained.

This method can further include administering to the individual a first vaccine composition and then at a later time, administering a second vaccine composition comprising a nanoparticle that comprises an HA-SS-ferritin fusion protein. The HA SS-ferritin fusion protein can comprise an amino acid sequence selected from SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98 or one that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical or at least 99% identical thereto, wherein the HA 55-ferritin fusion protein elicits an immune response to an influenza virus. The HA 55-ferritin fusion protein can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128, or one at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical or at least 99% identical thereto, wherein the HA 55-ferritin fusion protein elicits an immune response to an influenza virus.

In this method, the first vaccine composition can comprise a nanoparticle comprising an ectodomain from the hemagglutinin protein of an influenza virus selected from the group consisting of A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009 CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968 (1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo, B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris, H1), B/Brisbane/60/2008 (2008 Bris, B). Alternatively, the hemagglutinin of the first vaccine composition protein can comprise an amino acid sequence selected from SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38 or one that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 97% identical, at least about 99% identical thereto. Further, the first vaccine composition can comprise an HA-ferritin fusion protein comprising an amino acid sequence selected from SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68 or an amino acid sequence that is at least 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 97% identical, at least about 99% identical thereto, wherein the nanoparticle elicits an immune response against an influenza virus.

Administration of the boosting composition is generally weeks or months after administration of the priming composition.

A further embodiment of the present invention is a fusion protein comprising a monomeric ferritin subunit protein joined to an influenza hemagglutinin protein. The monomeric ferritin subunit protein can be selected from a bacterial ferritin, a plant ferritin, an algal ferritin, an insect ferritin, a fungal ferritin and a mammalian ferritin or can be a monomeric subunit of a Helicobacter pylori ferritin protein. The monomeric ferritin subunit protein can comprise a domain that allows the fusion protein to self-assemble into nanoparticles. In this embodiment, the monomeric ferritin subunit protein can comprise SEQ ID NO:2 or SEQ ID NO:5 or comprise at least 25 contiguous amino acids from or be at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 97% identical, at least about 99% to a sequence selected from SEQ ID NO:2 and SEQ ID NO:5 and the fusion protein can be capable of self-assembling into nanoparticles. Additionally, the monomeric subunit can comprise a region corresponding to amino acids 5-167 of SEQ ID NO:2.

In this embodiment, the hemagglutinin protein can comprise at least 25 amino acids from an influenza virus selected from A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009 CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968 (1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo, B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris, H1), and B/Brisbane/60/2008 (2008 Bris, B). Alternatively, the hemagglutinin protein can be capable of eliciting an immune response to a protein comprising an amino acid sequence selected from SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38 or one that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 97% identical, at least about 99% thereto.

In this embodiment, the fusion protein can comprise an amino acid sequence selected from SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68 or one that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 97% identical, at least about 99% thereto.

Further in this embodiment, the hemagglutinin protein can comprise a region selected from a region capable of allowing trimerization of the hemagglutinin protein, a stem region, an ectodomain, and a region comprising the amino acid sequence from the amino acid residue immediately distal to the last amino acid of the second helical coiled coil to the amino acid residue proximal to the first amino acid of the transmembrane domain. The hemagglutinin protein alternatively can comprise a region corresponding to amino acids 1-519 of SEQ ID NO:8, an amino acid sequence selected from the group consisting of amino acids 1-519 of SEQ ID NO:8 and SEQ ID NO:11, or a hemagglutinin spike domain. Further, the hemagglutinin protein can comprise the stem region from an influenza virus selected from A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009 CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968 (1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo, B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris, H1), or B/Brisbane/60/2008 (2008 Bris, B). The hemagglutinin protein can also comprise an amino acid sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 97% identical, at least about 99% to SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98.

In this embodiment, the fusion protein can comprise one or more linker sequences or an amino acid sequence of selected from the group consisting of SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128 or a sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 97% identical, at least about 99% thereto.

A further embodiment of the present invention is a nucleic acid molecule encoding any of the fusion proteins described above. In this embodiment, the nucleic acid molecule can be functionally linked to a promoter. Other embodiments of the invention include recombinant cells and viruses that comprise such nucleic acid molecules.

Another embodiment of the invention is a protein comprising an amino acid sequence at least 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 97% identical, at least about 99% to an amino acid selected from SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98, wherein the protein is joined to one or more trimerization domains. In this embodiment, the protein can be joined to at least a portion of the head region of an influenza hemagglutinin protein, comprise one or more linker regions or elicit an immune response against an influenza virus. A further embodiment is a nucleic acid molecule encoding such a protein.

BACKGROUND

Protective immune responses induced by vaccination against influenza virus are primarily directed to the viral hemagglutinin (HA) protein, which is a glycoprotein on the surface of the virus responsible for interaction of the virus with host cell receptors. HA proteins on the virus surface are trimers of hemagglutinin protein monomers that are enzymatically cleaved to yield amino-terminal HA1 and carboxy-terminal HA2 polypeptides. The globular head consists exclusively of the major portion of the HA1 polypeptide, whereas the stem that anchors the hemagglutinin protein into the viral lipid envelope is comprised of HA2 and part of HA1. The globular head of a hemagglutinin protein includes two domains: the receptor binding domain (RBD), an ˜148-amino acid residue domain that includes the sialic acid-binding site, and the vestigial esterase domain, a smaller ˜75-amino acid residue region just below the RBD. The top part of the RBD adjacent to the 2,6-sialic acid recognition sites includes a large region (amino acids 131-143, 170-182, 205-215 and 257-262, 1918 numbering) (referred to herein as the RBD-A region) of over 6000 A² per trimer that is 95% conserved between A/South Carolina/1/1918 (1918 SC) and A/California/04/2009 (2009 CA) pandemic strains. The globular head includes several antigenic sites that include immunodominant epitopes. Examples include the Sa, Sb, Ca₁, Ca₂ and Cb antigenic sites (see, for example, Caton A J et al, 1982, Cell 31, 417-427). The RBD-A region includes the Sa antigenic site and part of the Sb antigenic site.

Antibodies against influenza often target variable antigenic sites in the globular head of HA, which surround a conserved sialic acid binding site, and thus, neutralize only antigenically closely related viruses. The variability of the HA head is due to the constant antigenic drift of influenza viruses and is responsible for seasonal endemics of influenza. In contrast, gene segments of the viral genome can undergo reassortment (antigenic shift) in host species, creating new viruses with altered antigenicity that are capable of becoming pandemics [Salomon, R. et al. Cell 136, 402-410 (2009)]. Until now, each year, influenza vaccine is updated to reflect the predicted HA and neuraminidase (NA) for upcoming circulating viruses.

Current vaccine strategies for influenza use either a chemically inactivated or a live attenuated influenza virus. Both vaccines are generally produced in embryonated eggs which present major manufacturing limitations due to the time consuming process and limited production capacity. Another more critical limitation of current vaccines is its highly strain-specific efficacy. These challenges became glaring obvious during emergence of the 2009 H1N1 pandemic, thus validating the necessity for new vaccine platforms capable of overcoming these limitations. Virus-like particles represent one of such alternative approaches and are currently being evaluated in clinical trials [Roldao, A. et al. Expert Rev Vaccines 9, 1149-1176 (2010); Sheridan, C. Nat Biotechnol 27, 489-491 (2009)]. Instead of embryonated eggs, VLPs that often comprise HA, NA and matrix protein 1 (M1) can be mass-produced in mammalian or insect cell expression systems [Haynes, J. R. Expert Rev Vaccines 8, 435-445 (2009)]. The advantages of this approach are its particulate, multivalent nature and the authentic display of properly folded, trimeric HA spikes that faithfully mimic the infectious virion. In contrast, by the nature of its assembly, the enveloped VLPs contain a small but finite host cell component that may present potential safety, immunogenicity challenges following repeated use of this platform [Wu, C. Y. et al. PLoS One 5, e9784 (2010)]. Moreover, the immunity induced by the VLPs is essentially the same as current vaccines do, and thus, does not likely improve both potency and breadth of vaccine-induced protective immunity. In addition to VLPs, a recombinant HA protein has also been evaluated in humans [Treanor, J. J. et al. Vaccine 19, 1732-1737 (2001); Treanor, J. J. JAMA 297, 1577-1582 (2007)], though the ability to induce protective neutralizing antibody titers are limited. The recombinant HA proteins used in those trials were produced in insect cells and might not form native trimer preferentially [Stevens, J. Science 303, 1866-1870 (2004)].

Recently, entirely new classes of broadly neutralizing antibodies against influenza viruses were isolated. One class of antibodies recognizes the highly conserved HA stem [Corti, D. et al. J Clin Invest 120, 1663-1673 (2010); Ekiert, D. C. et al. Science 324, 246-251 (2009); Kashyap, A. K. et al. Proc Natl Acad Sci USA 105, 5986-5991 (2008); Okuno, Y. et al. J Virol 67, 2552-2558 (1993); Sui, J. et al. Nat Struct Mol Biol 16, 265-273 (2009); Ekiert, D. C. et al. Science 333, 843-850 (2011); Corti, D. et al. Science 333, 850-856 (2011)], and another class of antibodies precisely recognizes the sialic acid binding site of the RBD on the variable HA head [Whittle, J. R. et al. Proc Natl Acad Sci USA 108, 14216-14221 (2011); Krause, J. C. et al. J Virol 85, 10905-10908 (2011)]. Unlike strain-specific antibodies, those antibodies are capable of neutralizing multiple antigenically distinct viruses, and hence inducing such antibodies has been a focus of next generation universal vaccine [Nabel, G. J. et al. Nat Med 16, 1389-1391 (2010)]. However, robustly eliciting these antibodies with such heterologous neutralizing profile by vaccination has been difficult [Steel, J. et al. MBio 1, e0018 (2010); Wang, T. T. et al. PLoS Pathog 6, e1000796 (2010); Wei, C. J. et al. Science 329, 1060-1064 (2010)].

Despite several alternatives to conventional influenza vaccines, advances in biotechnology in past decades have allowed engineering of biological materials to be exploited for the generation of novel vaccine platforms. Ferritin, an iron storage protein found in almost all living organisms, is an example which has been extensively studied and engineered for a number of potential biochemical/biomedical purposes [Iwahori, K. U.S. Patent 2009/0233377 (2009); Meldrum, F. C. et al. Science 257, 522-523 (1992); Naitou, M. et al. U.S. Patent 2011/0038025 (2011); Yamashita, I. Biochim Biophys Acta 1800, 846-857 (2010)], including a potential vaccine platform for displaying exogenous epitope peptides [Carter, D. C. et al. U.S. Patent 2006/0251679 (2006); Li, C. Q. et al. Industrial Biotechnol 2, 143-147 (2006)]. Its use as a vaccine platform is particularly interesting because of its self-assembly and multivalent presentation of antigen which induces stronger B cell responses than monovalent form as well as induce T-cell independent antibody responses [Bachmann, M. F. et al. Annu Rev Immunol 15, 235-270 (1997); Dintzis, H. M. et al. Proc Natl Acad Sci USA 73, 3671-3675 (1976)]. Further, the molecular architecture of ferritin, which consists of 24 subunits assembling into an octahedral cage with 432 symmetry has the potential to display multimeric antigens on its surface.

There remains a need for an efficacious influenza vaccine that provides robust protection against influenza virus. There particularly remains a need for an influenza vaccine that protects individuals from heterologous strains of influenza virus, including evolving seasonal and pandemic influenza virus strains of the future. The present invention meets this need by providing a novel HA-ferritin nanoparticle (HA-ferritin np) influenza vaccine that is easily manufactured, potent, and elicits broadly neutralizing influenza antibodies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Molecular design and construction of ferritin particles displaying influenza virus hemagglutinin. (a) Ribbon diagram of a subunit of H. pylori nonheme ferritin (PDB: 3bve) (left). Amino- and carboxyl-termini are labeled as N and C, respectively. Three ferritin subunits surrounding an octahedral three-fold axis are shown as a ribbon diagram (middle). Residue Asp5 is indicated. The octahedral assembly of the ferritin particle (viewed at 8 Å resolution along an octahedral three-fold axis) and A/Solomon Islands/3/2006 (H1N1) HA trimer (PDB: 3sm5) (viewed down from membrane proximal side) (right). The measured average distance between the Asp5 residues in each ferritin subunit surrounding an octahedral three-fold axis is shown as a triangle. The same equilateral triangle (a=b=c=28 Å) is also drawn on the HA trimer (right). (b) Schematic representation of the HA-ferritin expression vector used for protein production. (c) Chromatogram of the size exclusion chromatography of ferritin nanoparticles (np) and HA-np (left). Molecular weights (kDa) of calibration standards are indicated above the curves with vertical lines. Calculated molecular weights for ferritin nanoparticles and HA-np were 419 and 2,165 kDa, respectively, and were within 10% of the predicted molecular weights (408 and 2,040 kDa, respectively). Particle size distribution (radius) of purified ferritin nanoparticles and HA-np was determined by dynamic light scattering (middle). Measured mean diameters (d) are indicated. The polydispersity indices of purified ferritin np and HA-np were 0.035 and 0.011, respectively. Purified HA trimer (thrombin uncleaved), HA-np and ferritin nanoparticles were analyzed by SDS-PAGE (right). (d) Negatively stained transmission electron microscopy images of ferritin nanoparticles (left) and HA-np (right). Images were originally recorded at 67,000× magnification. (e) Models representing octahedral four-, three- and two-fold symmetries of HA-ferritin np (top panels) and actual TEM image (bottom panels) are shown. Visible HA spikes are numbered in the images.

FIG. 2. Genetic and structural comparison of ferritins. (a) Phylogenetic tree analysis of ferritins found in RSCB PDB. Twenty-two sequences contain 16 ferritins including Vc (Vibrio cholerae), Ec (E. coli), Hp (H. pylori), Af (Archaeoglobus fulgidus), Pf (Pyrococcus furiosus), Tm (Thermatoga maritime), Pm (Pseudo-nitzschia multiseries), Tn (L) (Trichoplusia ni light chain), Soybean (chloroplastic), Horse (L) (light chain), Human (L), (H) and (M) (light, heavy chains and mitochondrial, respectively), Mouse (L) (light chain), and Frog (M) and (L) (middle and lower subunits, respectively), and 6 bacterioferritins (B) including Mt (B) (Mycobacterium tuberculosis), Pa (B) (Pseudomonas aeruginosa), Rs (B) (Rhodabacter sphaeroides), Bm (B) (Brucella melitensis), Av (B) (Azobacter vinelandii), and Ec (B). Protein sequences were aligned using Clustal W2 (www.ebi.ac.uk/Tools/msa/clustalw2) with Gonnet matrix and a phylogenetic tree was generated with the Phylodendron program (http://iubio.bio.indiana.edu/treeapp/treeprint-form.html) using the neighbor joining method. (b) Comparison of surface exposed residues between H. pylori and mouse (light chain) (left) or human (light chain) (middle), and mouse and human (light chains) (right). Conservation of surface exposed residues was rendered by UCSF Chimera using a protein sequence alignment generated by Clustal W2. Conserved and varied residues between the two ferritins are shown as light and dark residues, respectively. PDB files 3bve (H. pylori) (left and middle) and 1h96 (mouse light chain) (right) were used for surface rendering.

FIG. 3. Antigenic characterization of HA-ferritin np. (a) Binding of mAbs directed to globular head and stem of HA was measured by ELISA. Equal amount (200 ng of HA per well) of HA trimer (▭), TIV (▪), HA-ferritin () or Ferr (equimolar amount as HA-Ferr) (◯) were coated on the plates and wells were probed with anti-head mAb (3u-u) and anti-stem mAb CR6261. The half maximal effective concentrations (EC₅₀) of binding were calculated for each antibody and showed as ng ml⁻¹ (b) Inhibition of antibody-mediated neutralization of 1999 NC pseudotyped virus by using HA trimer, HA-Ferr or Ferr as a competitor. Inhibition of neutralization was plotted as percent inhibition respect to no competitor control. The anti-stem neutralizing mAbs, F10 (left) and CR6261 (right) were used at 3.125 and 25 μg ml⁻¹, respectively. Competitor proteins were added to the reactions at a final concentration of 20 μg ml⁻¹.

FIG. 4. Immune responses in HA-np-immunized mice. (a) HAI (left), IC₉₀ neutralization (middle), and anti-HA ab endpoint titers (right) against 1999 NC HA after two immunizations with 0.17 μg (amount of H1 HA) of TIV or HA-np with or without Ribi adjuvant and a 3-week interval. The immune sera were collected 2 weeks after the second immunization. The data are presented as box-and-whiskers plots (boxed from lower to upper quartile with whiskers from minimum to maximum) with lines at the mean (n=5). (b) Neutralization breadth of the immune sera elicited by HA-trimer, TIV, or HA-np. An additional group of mice (n=4) was immunized twice with 20 μg of trimeric HA protein using Ribi adjuvant and a 4-week interval. The immune sera were collected 2 weeks after the second immunization. IC₅₀ neutralization titers against a panel of H1N1 pseudotyped viruses were determined. (c) Cellular and humoral immune responses against H. pylori (top) and mouse (bottom) ferritins. Mice were immunized twice with 1.67 μg (amount of H1 HA) of TIV or HA-np, or 0.57 μg of ferritin nanoparticles (equimolar to HA-np) using Ribi adjuvant and a 3-week interval. The splenocytes and immune sera were harvested 11 days after the second immunization. Cytokine-producing CD4⁺ and CD8⁺ T cells were measured by ICS (left), and ab response was detected by ELISA (right). All cells expressing IFN-γ, TNFα, or IL-2 were identified as cytokine⁺ cells. The percentage of cytokine⁺ cells in CD4⁺ and CD8⁺ T cells that were activated in response to stimulation with specific peptides covering the entire H. pylori or mouse ferritins (heavy and light chains combined) were plotted. Recombinant H. pylori and purified mouse (liver) ferritins were used for detecting anti-ferritin ab responses. The data are presented as box-and-whiskers plots with lines at the mean (n=5).

FIG. 5. Successive immunization of HA-nanoparticles in mice. Mice were pre-immunized with 1.67 μg (amount of HA) of 2009 Perth (H3) HA-nanoparticles or 0.57 μg (equimolar to HA-nanoparticle) of empty ferritin nanoparticles at week 0 and then immunized with 1.67 μg (amount of HA) of 1999 NC (H1) HA-nanoparticles at week 3. Ribi was used as an adjuvant. Another group of mice was immunized with 1999 NC (H1) HA-nanoparticles without pre-immunization of empty ferritin nanoparticles or H3 HA-nanoparticles. (a) Ab responses to H. pylori ferritin (left) and 2009 Perth H3 HA (right). Immune sera collected 2 weeks after the immunization with H3 HA-nanoparticles or empty ferritin nanoparticles were analyzed by ELISA. (b) Immune responses to 1999 NC (H1) after 1999 NC (H1) HA-nanoparticle immunization. Naïve mice or mice with pre-immunity to ferritin or H3 HA were immunized with H1 HA-nanoparticles at week 3 and HAI (left), IC₉₀ neutralization (middle) and ELISA (right) Ab titers were measured 2 weeks after the immunization. The data are presented as box-and-whiskers plots with lines at the mean (n=5).

FIG. 6. Development of trivalent HA-np. (a) HA-np consisting of HAs from 2009 CA (H1), 2009 Perth (H3) or 2006 FL (type B) were purified and visualized by TEM. (b) HAI titers against 2009 CA (H1N1) and 2009 Perth (H3N2) viruses in immunized mice. Mice were immunized twice with 1.67 μg (amount of HA) of monovalent H1, monovalent H3, monovalent type B, or 5.0 μg (total amount of HA) of trivalent HA-np or TIV (2011-2012 season) using Ribi adjuvant with a 3-week interval. Immune sera were collected 2 weeks after the second immunization. The data are presented as box-and-whiskers plots with lines at the mean (n=5).

FIG. 7. Protective immunity induced in ferrets immunized with the HA-np. Ferrets were immunized twice with PBS (control), 7.5 ug (2.5 ug of H1 HA) of TIV or 2.5 ug (amount of HA) 1999 NC HA-np using Ribi adjuvant and a 4-week interval. Control animals received PBS. (a) HA1 (left), IC90 neutralization (middle), and anti-HA ab endpoint titers (right) in immunized ferrets against homologous 1999 NC HA were determined. Immune sera were collected 3 and 2 weeks after the first (R. Salomon, R. G. Webster, The influenza virus enigma. Cell 136, 402-410 (2009) and second (L. C. Lambert, A. S. Fauci, Influenza vaccines for the future. N Engl J Med 363, 2036-2044 (2010)) immunizations, respectively. The data are presented as box and whisker plots with lines at the mean (n=6). (b) Protection of immunized ferrets from an unmatched 2007 Bris virus challenge. Ferrets were challenged with 10^(6.5) 50% egg infectious dose (EID50) of 2007 Bris virus 5 weeks after the second immunization. Virus titers in the nasal washes from 1, 3 and 5 days post challenge were determined by a 50% tissue culture infectious dose (TCID₅₀) assay (left). One of six ferrets in the TIV-immunized group showed measurable virus on day 5. Virus titers in 4 out of 6 ferrets on day 3 and 6 out of 6 ferrets on day 5 in the HA-np-immunized group were under the detection limit (<102). The mean viral loads with standard deviation (s.d.) at each time point were plotted (n=6). Change in the body weight after the virus challenge was also monitored (right). Each data point represents the mean percent change in body weight from the pre-challenge (day 0). The mean body weight changes with standard error (s.e.) at each time point were plotted (n=6).

FIG. 8. Improved neutralization breadth and detection of stem- and RBS-directed abs. (a) Neutralization breadth of immune sera in ferrets. IC₅₀ neutralization titers against a panel of H1N1 pseudotyped viruses (left) and HAI titers against 1934 PR8 and 2007 Bris H1N1 viruses (right) were determined. The HAI titers are presented as box-and-whiskers plots with lines at the mean (n=6). (b) Stem- and RBS-directed abs elicited by HA-np immunization. Ferret immune sera (diluted 1:100) were pre-absorbed with ΔStem HA-expressing cells and their binding to WT or ΔStem HA were analyzed by ELISA (left). The immune sera (diluted 1:1,000) were pre-absorbed with ΔRBS HA-expressing cells and their binding to WT or ΔRBS HA were analyzed by ELISA (middle). The mean endpoint dilution titers were plotted with s.d. (n=6). Competition ELISA with stem-directed mAb CR6261 (right). The immune sera pre-absorbed with ΔStem were tested for binding to HA in the presence of an isotype control IgG or CR6261. Each symbol represents the titer of an individual ferret (n=6). (c) Neutralization competition with WT, ΔStem or ΔRBS HA protein (left). The neutralization of HA-np immune sera against 1986 Sing, 1995 Beijing, 1999 NC and 2007 Bris was measured in the presence of irrelevant protein (control), WT, ΔStem or ΔRBS HA as a competitor. Percent neutralizations at serum dilution 1:200 (1986 Sing, 2007 Bris), 1:800 (1995 Beijing) or 1:3,200 (1999 NC) were plotted. Each symbol represents the individual ferret serum and mean is indicated as a red line with s.d. (n=6 for 1986 Sing, 1995 Beijing and 1999 NC; n=3 for 2007 Bris). The relative contribution of the stem- and RBS-directed neutralization was determined by the inhibition of neutralization for each competitor protein (right). Mean percent contributions in neutralizing each virus were plotted as pile-up bars (n=6).

FIG. 9. Characterization of ΔRBS HA probe. (a) Crystal structure of HA (A/Solomon Islands/3/2006) complex with an anti-RBS mAb CH65 Fab (PDB: 3sm5) (J. R. Whittle et al., Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin. Proc Natl Acad Sci USA 108, 14216-14221 (2011)) (left). Close up view of CH65 contact area (right). The residue HA1 190 which has been mutated to be glycosylated in ΔRBS mutant is highlighted. The CH65 Fab-bound HA1 protomer is darkened. (b) Characterization of the soluble trimer of WT and ΔRBS HAs from 1999 NC and 2007 Bris. The WT and ΔRBS HA proteins were immunoprecipitated with anti-RBS (CH65), stem (CR6261) and control (anti-HIV, VRC01) mAbs. Immune complexes were then dissolved in Lamini buffer and analyzed by SDS-PAGE. Antibody heavy and light chains are labeled as HC and LC, respectively.

FIG. 10. Purification of HA-np. HA-np were purified by routine iodixanol gradient ultracentrifugation routinely. Fractions containing HA-np were confirmed by SDS-PAGE and Western blotting using a mAb against 1999 NC HA. The HA-np were enriched in the fraction with density of ˜1.15 g/ml.

FIG. 11. Protocol for immunization of mice and ferrets using pan-group 1 HA-ferritin np. Mice were injected intramuscularly twice (Week 0 and week 4) with PBS (control) or 6.8 ug (1.7 ug of each HA-ferritin np) pan-group 1 vaccine in Ribi. Ferrets were injected intramuscularly twice (Week 0 and week 4) with PBS (control) or 10 ug (2.5 ug of each HA-ferritin np) pan-group 1 vaccine in Ribi.

FIG. 12. Neutralization activity of mouse antisera against Group1 HA pseudotyped viruses. Neutralization activity of murine antisera from control or pan Group1 HA-np immunized mice against the indicated HA pseudotyped viruses. IC50 titers are shown for all panels.

FIG. 13. Neutralization activity of ferret antisera against Group1 HA pseudotyped viruses. Neutralization activity of ferret antisera from control or pan Group1 HA np immunized ferrets against the indicated HA pseudotyped viruses. IC50 titers are shown for all panels.

FIG. 14. H1 hemaggulutination inhbitions (HAI) assays were performed using the sera obtained from the ferritin immunization studies. These studies were performed using actual H1 virus, and H2 and H5 HAI were performed using HA-ferritin np.

FIG. 15. Protection of ferrets from viral challenge with Influenza A/Brisbane/59/2007 Brisbane (H1N1) (2007 Bris). Two groups of ferrets (n=6 for control and n=5 for pan-group1 immune) were immunized with pan Group1 HA np vaccine or PBS (control) and challenged with heterologous 2007 Bris virus (10^(6.5) EID₅₀). Virus titers were measured in nasal swabs collected on day 3 and day 5 post challenge. Titers were determined using end-point titration in MDCK cells.

FIG. 16. Protection of ferrets from viral challenge with Influenza A/Mexico/2009 (H1N1) (2009 Mex). Two groups of ferrets (n=6) were immunized with pan Group1 HA np vaccine or PBS (control) and challenged with heterologous 2009 Mex virus (10^(6.5) EID₅₀). Virus titers were measured in nasal swabs collected on day 3 and day 5 post challenge. Titers were determined using end-point titration in MDCK cells.

FIG. 17. Conservation of the influenza HA stem region. (left, right) Neutralizing antibodies that react with both Group 1 and Group 2 viruses act at the sites of vulnerability shown in the Figure. (Right) Space filling model of influenza HA protein illustrating amino acid sequence conservation in over 800 human H1N1 strains. Light residues indicate residues that are 100% conserved. Dark residues as indicate sites of variation.

FIG. 18. Design of HA Stabilized Stem protein. (A) Schematic of the HA SS (bottom) in comparison to HA (top). HA SS was constructed by inserting a GWG linker between residues 42 and 314 of HA1 RBD head, a gp41 post-fusion trimerization motif inserted in place of residues 59 through 93 of HA2, a GG linker between HA2 and the gp41 HR2 helix and an NGTGGGSG linker between the two gp41 helices. The gene sequence of H1 NC 99 SS is provided in the supplemental materials. (B) Trimeric and monomeric representation of HA (PDB entry 1RU7) in comparison to the HA SS model. Coloring is respective to above panel, with the monomeric representation also illustrating the CR6261 epitope as yellow and HA residues which are omitted in the stabilized HA stem as grey. (C) CR6261, FI6v3, and the germline of the VH1-69 Ab 70-5B03 have similar affinity to HA and SS by ELISA. HA SS competes with CR6261 (D) binding to HA and (E) neutralization of H1N1 pseudovirus similar to soluble HA trimer.

FIG. 19. Size exclusion chromatogram of HA and HA SS probes. Calibration standards are shown above the curves as vertical lines.

FIG. 20. Electron microscopic analysis of nanoparticles. Purified SS-np were subjected to transmission electron microscopic analysis. The samples were negatively stained with ammonium molybdate and images were recorded on a Tecnai T12 microscope (FEI) at 80 kV with a CCD camera (AMT Corp.). Images of lower (left) and higher (right) magnifications are shown. The SS spikes were protruding perpendicularly from the particle core and clearly visible.

FIG. 21. Antigenic characterization of HA SS-ferritin np. The ability of purified HA SS and HA SS-np to bind to monoclonal Abs CR6261 (left) and FI6v3 (right) was characterized by ELISA. HA and HIV gp120 proteins served as controls.

FIG. 22. Immune sera of mice immunized heterologously with HA-np prime and HA SS-np boost are reactive to the conserved HA stem epitope. Antibodies elicited by vaccination target the conserved HA stem epitope as individual mice possess differential binding (a minimum of 2-fold difference in endpoint dilution) between wt and Δstem HA variants. The percentage of mice displaying differential binding is given above matched wt and Δstem constructs. Error bars represent standard error.

FIG. 23. Immune sera of mice immunized with HA SS neutralizes diverse pseudovirus stains. IC₅₀ values are shown for individual mice against H1 homosubtypic strains and H2, H5 and H9 group-1 heterosubtypic strains. Dashed lines represents the lowest dilution assayed (50). Error bars represent standard error.

FIG. 24. Boosting with HA SS-np increases neutralizing titers in ferrets against H1N1 New Calendonia. Pseudovirus neutralizing titers were calculated for preimmune, HA FL-np primed, and HA SS-np boosted sera from individual mice. Error bars represent standard deviation of values.

FIG. 25. Map and sequence of CMV8x/R-H1NC HA(517)_SGG-egm and the related HA-ferritin fusion protein.

FIG. 26. Map and sequence of CMV8x/R-H1CA HA(518)_SGG-egm and the related HA-ferritin fusion protein.

FIG. 27. Map and sequence of CMV8x/R-H2Sing HA(514)_SGG-egm and the related HA-ferritin fusion protein.

FIG. 28. Map and sequence of CMV8x/R-H3HK HA(519)_SGG-egm and the related HA-ferritin fusion protein.

FIG. 29. Map and sequence of CMV8x/R-H3Bris HA(519)_SGG-egm and the related HA-ferritin fusion protein.

FIG. 30. Map and sequence of CMV8x/R-H5Indo HA(520)_SGG-egm and the related HA-ferritin fusion protein.

FIG. 31. Map and sequence of CMV8x/R-B.Florida HA(534)_SGG-egm and the related HA-ferritin fusion protein.

FIG. 32. Map and sequence of CMV8x/R-H3Perth HA(519)_SGG-egm and the related HA-ferritin fusion protein.

FIG. 33. Map and sequence of CMV8x/R-H1Bris HA(517)_SGG-egm and the related HA-ferritin fusion protein.

FIG. 34. Map and sequence of CMV8x/R-B.Bris HA(535)_SGG-egm and the related HA-ferritin fusion protein.

FIG. 35. Map and sequence of CMV8x/R—H1NC SS Gen4.55_SGG-egm and the related HA SS-ferritin fusion protein.

FIG. 36. Map and sequence of CMV/R H1 CA SS/Gen4.55/ferritin.

FIG. 37. Map and sequence of CMV/R H1 Bris SS/Gen4.55/ferritin.

FIG. 38. Map and sequence of CMV/R H2 Sing SS/Gen4.55/ferritin.

FIG. 39. Map and sequence of CMV/R H3 Bris SS/Gen4.55/ferritin.

FIG. 40. Map and sequence of CMV/R H3 Perth SS/Gen4.55/ferritin.

FIG. 41. Map and sequence of CMV/R H3 HK68 SS/Gen4.55/ferritin.

FIG. 42. Map and sequence of CMV/R H5 Indo SS/Gen4.55/ferritin.

FIG. 43. Map and sequence of CMV/R B Bris SS/Gen4.55/ferritin.

FIG. 44. Map and sequence of CMV/R B FL SS/Gen4.55/ferritin.

FIG. 45. Illustration showing hemagglutination reaction.

FIG. 46. Comparison of the hemagglutination activities of HA-ferritin nanoparticle and H5N1 vaccine. Inactivated vaccine (A) and HA-ferritin nanoparticles (B) were serially diluted in PBS and incubated with 0.5% chicken red blood cells (RBCs) for 30 minutes at room temperature. A series of dilutions were also performed using PBS as a control (C). Hemagglutination mediated through sialic acid moiety on RBC surface and the sialic acid-binding site on HA was determined by formation of lattice-structure of multiple RBCs (lack of red dot in the well).

FIG. 47. Mutation in sialic acid-binding site (ΔSA; Y98F) diminishes hemagglutination of HA-ferritin nanoparticles. Hemagglutination activity of HA-ferritin nanoparticles was diminished by a mutation in the sialic acid-binding site on HA.

FIG. 48. Titration of antibodies inhibiting hemagglutination using two assay systems (traditional HAI assay and nanoparticle-based HAI assay). Sera from twenty-four ferrets were pretreated with receptor-destroying enzyme (RDE) and heat inactivated were titrated by using A/New Calcdonia/20/99 (H1N1) virus or HA-np having the same HA. The samples include 6 negative (pre-immune) and 24 test (post-immune) sera. The titers are shown as scattered dots (left) and comparative (middle) plots. Correlation between the two assays was assessed (right).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel vaccine for influenza virus. More specifically, the present invention relates to novel, influenza hemagglutinin protein-based vaccines that elicit an immune response against a broad range of influenza viruses. It also relates to self-assembling ferritin-based, nanoparticles that display immunogenic portions of influenza hemagglutinin protein on their surface. Such nanoparticles are useful for vaccinating individuals against influenza virus. Accordingly, the present invention also relates to fusion proteins for producing such nanoparticles and nucleic acid molecules encoding such proteins. Additionally, the present invention relates to, methods of producing nanoparticles of the present invention, and methods of using such nanoparticles to vaccinate individuals.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the claims.

It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. As such, the terms “a”, “an”, “one or more” and “at least one” can be used interchangeably. Similarly the terms “comprising”, “including” and “having” can be used interchangeably. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

In addition to the above, unless specifically defined otherwise, the following terms and phrases, which are common to the various embodiments disclosed herein, are defined as follows:

As used herein, the term immunogenic refers to the ability of a specific protein, or a specific region thereof, to elicit an immune response to the specific protein, or to proteins comprising an amino acid sequence having a high degree of identity with the specific protein. According to the present invention, two proteins having a high degree of identity have amino acid sequences at least 80% identical, at least 85% identical, at least 87% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical or at least 99% identical.

As used herein, an immune response to a vaccine, or nanoparticle, of the present invention is the development in a subject of a humoral and/or a cellular immune response to a hemagglutinin protein present in the vaccine. For purposes of the present invention, a “humoral immune response” refers to an immune response mediated by antibody molecules, including secretory (IgA) or IgG molecules, while a “cellular immune response” is one mediated by T-lymphocytes and/or other white blood cells. One important aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells (“CTL”s). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes, or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A cellular immune response also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells.

Thus, an immunological response may be one that stimulates CTLs, and/or the production or activation of helper T-cells. The production of chemokines and/or cytokines may also be stimulated. The vaccine may also elicit an antibody-mediated immune response. Hence, an immunological response may include one or more of the following effects: the production of antibodies (e.g., IgA or IgG) by B-cells; and/or the activation of suppressor, cytotoxic, or helper T-cells and/or T-cells directed specifically to a hemagglutinin protein present in the vaccine. These responses may serve to neutralize infectivity, and/or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection to an immunized individual. Such responses can be determined using standard immunoassays and neutralization assays, well known in the art.

According to the present invention all nomenclature used to classify influenza virus is that commonly used by those skilled in the art. Thus, a Type, or Group, of influenza virus refers to influenza Type A, influenza Type B or influenza type C. It is understood by those skilled in the art that the designation of a virus as a specific Type relates to sequence difference in the respective M1 (matrix) protein or NP (nucleoprotein). Type A influenza viruses are further divided into Group1 and Group 2. These Groups are further divided into subtypes, which refers to classification of a virus based on the sequence of its HA protein. Examples of current commonly recognized subtypes are H1, H2, H3, H4, H5, H6, H7, H8, H8, H10, H11, H12, H13, H14, H15 or H16. Group 1 influenza subtypes are H1, H2, H5, H7 and H9. Group 2 influenza subtypes are H4, H4, H6, H8, H10, H11, H12, H13, H14, H15 and H16. Finally, the term strain refers to viruses within a subtype that differ from one another in that they have small, genetic variations in their genome.

As used herein, neutralizing antibodies are antibodies that prevent influenza virus from completing one round of replication. As defined herein, one round of replication refers the life cycle of the virus, starting with attachment of the virus to a host cell and ending with budding of newly formed virus from the host cell. This life cycle includes, but is not limited to, the steps of attaching to a cell, entering a cell, cleavage and rearrangement of the HA protein, fusion of the viral membrane with the endosomal membrane, release of viral ribonucleoproteins into the cytoplasm, formation of new viral particles and budding of viral particles from the host cell membrane.

As used herein, broadly neutralizing antibodies are antibodies that neutralize more than one type, subtype and/or strain of influenza virus. For example, broadly neutralizing antibodies elicited against an HA protein from a Type A influenza virus may neutralize a Type B or Type C virus. As a further example, broadly neutralizing antibodies elicited against an HA protein from Group I influenza virus may neutralize a Group 2 μm. AS an additional example, broadly neutralizing antibodies elicited against an HA protein from one sub-type or strain of virus, may neutralize another sub-type or strain of virus. For example, broadly neutralizing antibodies elicited against an HA protein from an H1 influenza virus may neutralize viruses from one or more sub-types selected from the group consisting of H2, H3, H4, H5, H6, H7, H8, H8, H10, H11, H12, H13, H14, H15 or H16.

As used herein, an influenza hemagglutinin protein, or HA protein, refers to a full-length influenza hemagglutinin protein or any portion thereof, that is capable of eliciting an immune response. Preferred HA proteins are those that are capable of forming a trimer. An epitope of a full-length influenza hemagglutinin protein refers to a portion of such protein that can elicit a neutralizing antibody response against the homologous influenza strain, i.e., a strain from which the HA is derived. In some embodiments, such an epitope can also elicit a neutralizing antibody response against a heterologous influenza strain, i.e., a strain having an HA that is not identical to that of the HA of the immunogen.

With regard to hemagglutinin proteins, it is understood by those skilled in the art that hemagglutinin proteins from different influenza viruses may have different lengths due to mutations (insertions, deletions) in the protein. Thus, reference to a corresponding region refers to a region of another proteins that is identical, or nearly so (e.g., at least 95%, identical, at least 98% identical or at least 99% identical), in sequence, structure and/or function to the region being compared. For example, with regard to the stem region of a hemagglutinin protein, the corresponding region in another hemagglutinin protein may not have the same residue numbers, but will have a nearly identical sequence and will perform the same function. To better clarify sequences comparisons between viruses, numbering systems are used by those in the field, which relate amino acid positions to a reference sequence. Thus, corresponding amino acid residues in hemagglutinin proteins from different strains of influenza may not have the same residue number with respect to their distance from the n-terminal amino acid of the protein. For example, using the H3 numbering system, reference to residue 100 in A/New Calcdonia/20/1999 (1999 NC, H1) does not mean it is the 100^(th) residue from the N-terminal amino acid. Instead, residue 100 of A/New Calcdonia/20/1999 (1999 NC, H1) aligns with residue 100 of influenza H3N2 strain. The use of such numbering systems is understood by those skilled in the art. Unless otherwise noted, reference to amino acids in hemagglutinin proteins herein is made using the H3 numbering system.

According to the present invention, a trimerization domain is a series of amino acids that when joined (also referred to as fused) to a protein or peptide, allow the fusion protein to interact with other fusion proteins containing the trimerization domain, such that a trimeric structure is formed. Any known trimerization domain can be used in the present invention. Examples of trimerization domains include, but are not limited to, the HIV-1 gp41 trimerization domain, the SIV gp41 trimerization domain, the Ebola virus gp-2 trimerization domain, the HTLV-1 gp-21 trimerization domain, the T4 fibritin trimerization domain (i.e., foldon), the yeast heat shock transcription factor trimerization domain, and the human collagen trimerization domain.

As used herein, a variant refers to a protein, or nucleic acid molecule, the sequence of which is similar, but not identical to, a reference sequence, wherein the activity of the variant protein (or the protein encoded by the variant nucleic acid molecule) is not significantly altered. These variations in sequence can be naturally occurring variations or they can be engineered through the use of genetic engineering technique know to those skilled in the art. Examples of such techniques are found in Sambrook J, Fritsch E F, Maniatis T et al., in Molecular Cloning—A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, pp. 9.31-9.57), or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, both of which are incorporated herein by reference in their entirety.

With regard to variants, any type of alteration in the amino acid, or nucleic acid, sequence is permissible so long as the resulting variant protein retains the ability to elicit neutralizing antibodies against an influenza virus. Examples of such variations include, but are not limited to, deletions, insertions, substitutions and combinations thereof. For example, with regard to proteins, it is well understood by those skilled in the art that one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10), amino acids can often be removed from the amino and/or carboxy terminal ends of a protein without significantly affecting the activity of that protein. Similarly, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids can often be inserted into a protein without significantly affecting the activity of the protein.

As noted, variant proteins of the present invention can contain amino acid substitutions relative to the influenza HA proteins disclosed herein. Any amino acid substitution is permissible so long as the activity of the protein is not significantly affected. In this regard, it is appreciated in the art that amino acids can be classified into groups based on their physical properties. Examples of such groups include, but are not limited to, charged amino acids, uncharged amino acids, polar uncharged amino acids, and hydrophobic amino acids. Preferred variants that contain substitutions are those in which an amino acid is substituted with an amino acid from the same group. Such substitutions are referred to as conservative substitutions.

Naturally occurring residues may be divided into classes based on common side chain properties:

1) hydrophobic: Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr;

3) acidic: Asp, Glu;

4) basic: Asn, Gln, His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.

In making amino acid changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. The hydropathic indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5). The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte et al., 1982, J. Mol. Biol. 157:105-31). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functionally equivalent protein or peptide thereby created is intended for use in immunological invention, as in the present case. The greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); and tryptophan (−3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity.

Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the HA protein, or to increase or decrease the immunogenicity, solubility or stability of the HA proteins described herein. Exemplary amino acid substitutions are shown below in Table 1.

TABLE 1 Amino Acid Substitutions Original Amino Acid Exemplary Substitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln, Lys, Arg Ile Leu, Val, Met, Ala Leu Ile, Val, Met, Ala Lys Arg, Gln, Asn Met Leu, Phe, Ile Phe Leu, Val, Ile, Ala, Tyr Pro Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr, Phe Tyr Trp, Phe, Thr, Ser Val Ile, Met, Leu, Phe, Ala

As used herein, the phrase significantly affect a proteins activity refers to a decrease in the activity of a protein by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%. With regard to the present invention, such an activity may be measured, for example, as the ability of a protein to elicit neutralizing antibodies against an influenza virus. Such activity may be measured by measuring the titer of such antibodies against influenza virus, or by measuring the number of types, subtypes or strains neutralized by the elicited antibodies. Methods of determining antibody titers and methods of performing virus neutralization assays are known to those skilled in the art. In addition to the activities described above, other activities that may be measured include the ability to agglutinate red blood cells and the binding affinity of the protein for a cell. Methods of measuring such activities are known to those skilled in the art.

As used herein, a fusion protein is a recombinant protein containing amino acid sequence from at least two unrelated proteins that have been joined together, via a peptide bond, to make a single protein. The unrelated amino acid sequences can be joined directly to each other or they can be joined using a linker sequence. As used herein, proteins are unrelated, if their amino acid sequences are not normally found joined together via a peptide bond in their natural environment(s) (e.g., inside a cell). For example, the amino acid sequences of monomeric subunits that make up ferritin, and the amino acid sequences of influenza hemagglutinin proteins are not normally found joined together via a peptide bond.

The terms individual, subject, and patient are well-recognized in the art, and are herein used interchangeably to refer to any human or other animal susceptible to influenza infection. Examples include, but are not limited to, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, seals, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The terms individual, subject, and patient by themselves, do not denote a particular age, sex, race, and the like. Thus, individuals of any age, whether male or female, are intended to be covered by the present disclosure and include, but are not limited to the elderly, adults, children, babies, infants, and toddlers. Likewise, the methods of the present invention can be applied to any race, including, for example, Caucasian (white), African-American (black), Native American, Native Hawaiian, Hispanic, Latino, Asian, and European. An infected subject is a subject that is known to have influenza virus in their body.

As used herein, a vaccinated subject is a subject that has been administered a vaccine that is intended to provide a protective effect against an influenza virus.

As used herein, the terms exposed, exposure, and the like, indicate the subject has come in contact with a person of animal that is known to be infected with an influenza virus.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

According to the present invention, vaccines are provided that elicit a broad immune response against influenza viruses. Some vaccines disclosed herein may elicit an immune response against the entire HA protein, while others may elicit an immune response against a specific region or portion of an influenza HA protein. Moreover, the inventors have discovered that specific fusion proteins comprising portions of hemagglutinin protein are useful for eliciting a broad immune response against influenza viruses. Each of these embodiments will now be disclosed in detail below.

Vaccines Against the Stem Region of Influenza HA Protein

As stated previously, the amino acid sequence of the stem region of the hemagglutinin protein is highly conserved across types, sub-types and strains of influenza viruses and contains a site of vulnerability for group 1 viruses. Thus, an immune response directed this region of the HA protein may protect individuals against influenza viruses from several types, sub-types and/or strains.

Consequently, one embodiment of the present invention is a protein that elicits an immune response against the stem region of an influenza HA protein. In one embodiment, the immune response can be directed against the stem region of an HA protein from a virus selected from the group consisting of influenza A viruses, influenza B viruses and influenza C viruses. In one embodiment, the immune response can be directed against the stem region of an HA protein from a virus selected from the group consisting of an H1 influenza virus, an H2 influenza virus, an influenza H3 virus, an influenza H4 virus, an influenza H5 virus, an influenza H6 virus, an H7 influenza virus, an H8 influenza virus, an H9 influenza virus, an H10 influenza virus, an H11 influenza virus, an H12 influenza virus, an H13 influenza virus, an H14 influenza virus, an H15 influenza virus and an H16 influenza virus. In one embodiment, the immune response can be directed against the stem region of an HA protein from a strain of virus selected from the group of viruses listed in Table 2.

TABLE 2 SEQ ID NO Comments FERRITIN 1 Coding sequence for ferritin monomeric subunit protein from H. pylori 2 Amino acid sequence encoded by SEQ ID NO: 1 3 Complement of SEQ ID NO1 4 Nucleic acid sequence encoding amino acids 5-167 from SEQ ID NO: 2; Asn19 has been replaced with Gln 5 Amino acid sequence encoded by SEQ ID NO: 3 6 Complement of SEQ ID NO3 FULL LENGTH HA 7 Nucleic acid sequence encoding full length hemagglutinin protein from A/New Caledonia/20/1999 (1999 NC, H1)(GenBank: AY289929) 8 Amino acid sequence encoded by SEQ ID NO: 7 (full length hemagglutinin protein from A/New Caledonia/20/1999 (1999 NC, H1)(GenBank: AY289929)) 9 Complement of SEQ ID NO: 7 ECTODOMAINS 10 Nucleic acid sequence encoding ectodomain from hemagglutinin protein from A/New Caledonia/20/1999 (1999 NC, H1). 11 Amino acid sequence encoded by SEQ ID NO: 10 (ectodomain from hemagglutinin protein from A/New Caledonia/20/1999 (1999 NC, H1). Amino acids 1-517 from SEQ ID NO: 8. 12 Complement of SEQ ID NO: 10 13 Nucleic acid sequence encoding ectodomain from hemagglutinin protein from A/California/04/2009 (2009 CA, H1). 14 Amino acid sequence encoded by SEQ ID NO: 13 (ectodomain from hemagglutinin protein from A/California/04/2009 (2009 CA, H1)) 15 Complement of SEQ ID NO: 13 16 Nucleic acid sequence encoding ectodomain from hemagglutinin protein from A/Singapore/1/1957 (1957 Sing, H2). 17 Amino acid sequence encoded by SEQ ID NO: 16 (ectodomain from hemagglutinin protein from A/Singapore/1/1957 (1957 Sing, H2)) 18 Complement of SEQ ID NO: 16 19 Nucleic acid sequence encoding ectodomain from hemagglutinin protein from A/Hong Kong/1/1968 (1968 HK, H3). 20 Amino acid sequence encoded by SEQ ID NO: 19) ectodomain from hemagglutinin protein from A/Hong Kong/1/1968 (1968 HK, H3)) 21 Complement of SEQ ID NO: 19 22 Nucleic acid sequence encoding ectodomain from hemagglutinin protein from A/Brisbane/10/2007 (2007 Bris, H3). 23 Amino acid sequence encoded by SEQ ID NO: 22 (ectodomain from hemagglutinin protein from A/Brisbane/10/2007 (2007 Bris, H3)) 24 Complement of SEQ ID NO: 22. 25 Nucleic acid sequence encoding ectodomain from hemagglutinin protein from A/Indonesia/05/2005 (2005 Indo, H5) 26 Amino acid sequence encoded by SEQ ID NO: 25 (ectodomain from hemagglutinin protein from A/Indonesia/05/2005 (2005 Indo, H5)) 27 Complement of SEQ ID NO: 25 28 Nucleic acid sequence encoding ectodomain from hemagglutinin protein from B/Florida/4/2006 (2006 Flo, B) 29 Amino acid sequence encoded by SEQ ID NO: 28 (ectodomain from hemagglutinin protein from B/Florida/4/2006 (2006 Flo, B)) 30 Complement of SEQ ID NO: 28 31 Nucleic acid sequence encoding ectodomain from hemagglutinin protein from A/Perth/16/2009 (2009 Per, H3) 32 Amino acid sequence encoded by SEQ ID NO: 31 (ectodomain from hemagglutinin protein from A/Perth/16/2009 (2009 Per, H3)) 33 Complement of SEQ ID NO: 31 34 Nucleic acid sequence encoding ectodomain from hemagglutinin protein from A/Brisbane/59/2007 (2007 Bris, H1) 35 Amino acid sequence encoded by SEQ ID NO: 34 (ectodomain from hemagglutinin protein from A/Brisbane/59/2007 (2007 Bris, H1)) 36 Complement of SEQ ID NO: 34 37 Nucleic acid sequence encoding ectodomain from hemagglutinin protein from B/Brisbane/60/2008 (2008 Bris, B) 38 Amino acid sequence encoded by SEQ ID NO: 37 (ectodomain from hemagglutinin protein from B/Brisbane/60/2008 (2008 Bris, B)) 39 Complement of SEQ ID NO: 37 FERRITIN-HA ECTODOMAIN FUSION 40 Nucleic acid sequence encoding SEQ ID NO: 41 41 Amino acid sequence of ferritin-HA fusion (ectodomain from hemagglutinin protein from A/New Caledonia/20/1999 (1999 NC, H1)) 42 Complement of SEQ ID NO: 40 43 Nucleic acid sequence encoding SEQ ID NO: 44 44 Amino acid sequence of ferritin-HA fusion (ectodomain from hemagglutinin protein from A/California/04/2009 (2009 CA, H1)) 45 Complement of SEQ ID NO: 43 46 Nucleic acid sequence encoding SEQ ID NO: 47 47 Amino acid sequence of ferritin-HA fusion (ectodomain from hemagglutinin protein from A/Singapore/1/1957 (1957 Sing, H2)) 48 Complement of SEQ ID NO: 46 49 Nucleic acid sequence encoding SEQ ID NO: 50 50 Amino acid sequence of ferritin-HA fusion (ectodomain from hemagglutinin protein from A/Hong Kong/1/1968 (1968 HK, H3)) 51 Complement of SEQ ID NO: 49 52 Nucleic acid sequence encoding SEQ ID NO: 53 53 Amino acid sequence of ferritin-HA fusion (ectodomain from hemagglutinin protein from A/Brisbane/10/2007 (2007 Bris, H3)) 54 Complement of SEQ ID NO: 52 55 Nucleic acid sequence encoding SEQ ID NO: 56 56 Amino acid sequence of ferritin-HA fusion (ectodomain from hemagglutinin protein from A/Indonesia/05/2005 (2005 Indo, H5)) 57 Compliment of SEQ ID NO: 55 58 Nucleic acid sequence encoding SEQ ID NO: 59 59 Amino acid sequence of ferritin-HA fusion protein (ectodomain from hemagglutinin protein from B/Florida/4/2006 (2006 Flo, B)) 60 Complement of SEQ ID NO: 58 61 Nucleic acid sequence encoding SEQ ID NO: 62 62 Amino acid sequence of ferritin-HA fusion protein (ectodomain from hemagglutinin protein from A/Perth/16/2009 (2009 Per, H3)) 63 Complement of SEQ ID NO: 61 64 Nucleic acid sequence encoding SEQ ID NO: 65 65 Amino acid sequence of ferritin-HA fusion protein (ectodomain from hemagglutinin protein from A/Brisbane/59/2007 (2007 Bris, H1)) 66 Complement of SEQ ID NO: 64 67 Nucleic acid sequence encoding SEQ ID NO: 68 68 Amino acid sequence of ferritin-HA fusion protein (ectodomain from hemagglutinin protein from B/Brisbane/60/2008 (2008 Bris, B)) 69 Complement of SEQ ID NO: 67 STEM REGION 70 Nucleic acid molecule encoding SEQ ID NO: 71 71 Amino acid sequence of stem region from A/New Caledonia/20/1999 (1999 NC, H1)(GenBank: AY289929) 72 Complement of SEQ ID NO: 70 73 Nucleic acid sequence encoding SEQ ID NO: 74 74 Amino acid sequence of stem region from A/California/04/2009 (2009 CA, H1) 75 Complement of SEQ ID NO: 73 76 Nucleic acid sequence encoding SEQ ID NO: 77 77 Amino acid sequence of stem region from A/Singapore/1/1957 (1957 Sing, H2) 78 Complement of SEQ ID NO: 76 79 Nucleic acid sequence encoding SEQ ID NO: 80 80 Amino acid sequence of stem region from A/Hong Kong/1/1968 (1968 HK, H3) 81 Complement of SEQ ID NO: 79 82 Nucleic acid sequence encoding SEQ ID NO: 83 83 Amino acid sequence of stem region from A/Brisbane/10/2007 (2007 Bris, H3) 84 Complement of SEQ ID NO: 82 85 Nucleic acid sequence encoding SEQ ID NO: 86 86 Amino acid sequence of stem region from A/Indonesia/05/2005 (2005 Indo, H5) 87 Complement of SEQ ID NO: 85 88 Nucleic acid sequence encoding SEQ ID NO: 89 89 Amino acid sequence of stem region from B/Florida/4/2006 (2006 Flo, B) 90 Complement of SEQ ID NO: 88 91 Nucleic acid sequence encoding SEQ ID NO: 92 92 Amino acid sequence of stem region from A/Perth/16/2009 (2009 Per, H3) 93 Complement of SEQ ID NO: 91 94 Nucleic acid sequence encoding SEQ ID NO: 95 95 Amino acid sequence of stem region from A/Brisbane/59/2007 (2007 Bris, H1) 96 Complement of SEQ ID NO: 94 97 Nucleic acid sequence encoding SEQ ID NO: 98 98 Amino acid sequence of stem region from B/Brisbane/60/2008 (2008 Bris, B) 99 Complement of SEQ ID NO: 97 FERRITIN-HA STEM REGION FUSION 100 Nucleic acid sequence encoding SEQ ID NO: 101 101 Amino acid sequence of ferritin-HA stem region fusion protein A/New Caledonia/20/1999 (1999 NC, H1) 102 Complement of SEQ ID NO: 100 103 Nucleic acid sequence encoding SEQ ID NO: 104 104 Amino acid sequence of ferritin-HA stem region fusion protein (H1 CA) 105 Complement of SEQ ID NO: 103 106 Nucleic acid sequence encoding SEQ ID NO: 107 107 Amino acid sequence of ferritin-HA stem region fusion protein (H2 Sing) 108 Complement of SEQ ID NO: 106 109 Nucleic acid sequence encoding SEQ ID NO: 110 110 Amino acid sequence of ferritin-HA stem region fusion protein (H3 Hong Kong) 111 Complement of SEQ ID NO: 109 112 Nucleic acid sequence encoding SEQ ID NO: 113 113 Amino acid sequence of ferritin-HA stem region fusion protein (H5 Indonesia) 114 Complement of SEQ ID NO: 112 115 Nucleic acid sequence encoding SEQ ID NO: 116 116 Amino acid sequence of ferritin-HA stem region fusion protein (A/Brisbane/59/2007 (2007 Bris, H1)) 117 Complement of SEQ ID NO: 115 118 Nucleic acid sequence encoding SEQ ID NO: 119 119 Amino acid sequence of ferritin-HA stem region fusion protein (A/Brisbane/10/2007 (2007 Bris, H3)) 120 Complement of SEQ ID NO: 118 121 Nucleic acid sequence encoding SEQ ID NO: 122 122 Amino acid sequence of ferritin-HA stem region fusion protein (A/Perth/16/2009 (2009 Per, H3)) 123 Complement of SEQ ID NO: 121 124 Nucleic acid sequence encoding SEQ ID NO: 125 125 Amino acid sequence of ferritin-HA stem region fusion protein (B/Brisbane/60/2008 (2008 Bris, B) 126 Complement of SEQ ID NO: 124 127 Nucleic acid sequence encoding SEQ ID NO: 128 128 Amino acid sequence of ferritin-HA stem region fusion protein (B/Florida/4/2006 (2006 Flo, B)) 129 Complement of SEQ ID NO: 127

One type of immune response is a B-cell response, which results in the production of antibodies against the antigen that elicited the immune response. Thus, one embodiment of the present invention is a protein that elicits antibodies that bind to the stem region of influenza HA protein from a virus selected from the group consisting of influenza A viruses, influenza B viruses and influenza C viruses. One embodiment of the present invention is a protein that elicits antibodies that bind to the stem region of influenza HA protein selected from the group consisting of an H1 influenza virus HA protein, an H2 influenza virus HA protein, an influenza H3 virus HA protein, an influenza H4 virus HA protein, an influenza H5 virus HA protein, an influenza H6 virus HA protein, an H7 influenza virus HA protein, an H8 influenza virus HA protein, an H9 influenza virus HA protein, an H10 influenza virus HA protein HA protein, an H11 influenza virus HA protein, an H12 influenza virus HA protein, an H13 influenza virus HA protein, an H14 influenza virus HA protein, an H15 influenza virus HA protein and an H16 influenza virus HA protein. One embodiment of the present invention is a protein that elicits antibodies that bind to the stem region of influenza HA protein from a strain of virus selected from the viruses listed in Table 2.

While all antibodies are capable of binding to the antigen which elicited the immune response that resulted in antibody production, preferred antibodies are those that neutralize an influenza virus. Thus, one embodiment of the present invention is a protein that elicits neutralizing antibodies that bind to the stem region of influenza HA protein from a virus selected from the group consisting of influenza A viruses, influenza B viruses and influenza C viruses. One embodiment of the present invention is a protein that elicits neutralizing antibodies that bind to the stem region of influenza HA protein selected from the group consisting of an H1 influenza virus HA protein, an H2 influenza virus HA protein, an influenza H3 virus HA protein, an influenza H4 virus HA protein, an influenza H5 virus HA protein, an influenza H6 virus HA protein, an H7 influenza virus HA protein, an H8 influenza virus HA protein, an H9 influenza virus HA protein, an H10 influenza virus HA protein HA protein, an H11 influenza virus HA protein, an H12 influenza virus HA protein, an H13 influenza virus HA protein, an H14 influenza virus HA protein, an H15 influenza virus HA protein and an H16 influenza virus HA protein. One embodiment of the present invention is a protein that elicits neutralizing antibodies that bind to the stem region of influenza HA protein from a strain of virus selected from the viruses listed in Table 2. One embodiment of the present invention is a protein that elicits neutralizing antibodies that bind to a protein comprising an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98. One embodiment of the present invention is a protein that elicits neutralizing antibodies that bind to a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98.

Neutralizing antibodies elicited by proteins of the present invention can neutralize viral infections by affecting any step in the life cycle of the virus. For example, neutralizing antibodies may prevent an influenza virus from attaching to a cell, entering a cell, releasing viral ribonucleoproteins into the cytoplasm, forming new viral particles in the infected cell and budding new viral particles from the infected host cell membrane. In one embodiment, neutralizing antibodies elicited by proteins of the present invention prevent influenza virus from attaching to the host cell. In one embodiment, neutralizing antibodies elicited by proteins of the present invention prevent influenza virus from entering the host cell. In one embodiment, neutralizing antibodies elicited by proteins of the present invention prevent fusion of viral membranes with endosomal membranes. In one embodiment, neutralizing antibodies elicited by proteins of the present invention prevent release of ribonucleoproteins into the cytoplasm of the host cell. In one embodiment, neutralizing antibodies elicited by proteins of the present invention prevent assembly of new virus in the infected host cell. In one embodiment, neutralizing antibodies elicited by proteins of the present invention prevent release of newly formed virus from the infected host cell.

Because the amino acid sequence of the stem region of influenza virus is highly conserved, neutralizing antibodies elicited by proteins of the present invention may be broadly neutralizing. That is, neutralizing antibodies elicited by proteins of the present invention may neutralize influenza viruses of more than one type, subtype and/or strain. Thus, one embodiment of the present invention is a protein that elicits broadly neutralizing antibodies that bind the stem region of influenza HA protein. One embodiment is a protein that elicits antibodies that bind the stem region of an HA protein from more than one type of influenza virus selected from the group consisting of influenza type A viruses, influenza type B viruses and influenza type C viruses. One embodiment is a protein that elicits antibodies that bind the stem region of an HA protein from more than one sub-type of influenza virus selected from the group consisting of an H1 influenza virus, an H2 influenza virus, an influenza H3 virus, an influenza H4 virus, an influenza H5 virus, an influenza H6 virus, an H7 influenza virus, an H8 influenza virus, an H9 influenza virus, an H10 influenza virus, an H11 influenza virus, an H12 influenza virus, an H13 influenza virus, an H14 influenza virus, an H15 influenza virus and an H16 influenza virus. One embodiment is a protein that elicits antibodies that bind the stem region of an HA protein from more than strain of influenza virus. One embodiment is a protein that elicits antibodies that bind the stem region of an HA protein from more than one strain of influenza virus selected from the viruses listed in Table 2. One embodiment of the present invention is a protein that elicits antibodies that bind more than one protein comprising an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98. One embodiment of the present invention is a protein that elicits neutralizing antibodies that bind to more than one protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98.

Particularly useful proteins of the present invention are those comprising an immunogenic portion of an influenza HA protein. Thus, one embodiment of the present invention is a protein comprising at least one immunogenic portion from the stem region of influenza HA protein, wherein the protein elicits neutralizing antibodies against an influenza virus. Such a protein is referred to as a stem-region protein (or a stem-region immunogen). One embodiment of the present invention is a protein comprising at least one immunogenic portion from the stem region of an HA protein from a virus selected from the group consisting of influenza type A viruses, influenza type B viruses and influenza type C viruses, wherein the protein elicits neutralizing antibodies against an influenza virus. One embodiment of the present invention is a protein comprising at least one immunogenic portion from the stem region of an HA protein selected from the group consisting of an H1 influenza virus HA protein, an H2 influenza virus HA protein, an influenza H3 virus HA protein, an influenza H4 virus HA protein, an influenza H5 virus HA protein, an influenza H6 virus HA protein, an H7 influenza virus HA protein, an H8 influenza virus HA protein, an H9 influenza virus HA protein, an H10 influenza virus HA protein HA protein, an H11 influenza virus HA protein, an H12 influenza virus HA protein, an H13 influenza virus HA protein, an H14 influenza virus HA protein, an H15 influenza virus HA protein and an H16 influenza virus HA protein. One embodiment of the present invention is a protein comprising at least one immunogenic portion from the stem region of an HA protein from the viruses listed in Table 2. One embodiment of the present invention is a protein comprising at least one immunogenic portion from a protein comprising an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98. One embodiment of the present invention is a protein comprising at least one immunogenic portion from a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98. In one embodiment, such proteins comprising immunogenic portions of the HA protein elicit the production of broadly neutralizing antibodies against influenza virus.

Immunogenic portions of proteins comprise epitopes, which are clusters of amino acid residues that are recognized by the immune system, thereby eliciting an immune response. Such epitopes may consist of contiguous amino acids residues (i.e., amino acid residues that are adjacent to one another in the protein), or they may consist of non-contiguous amino acid residues (i.e., amino acid residues that are not adjacent one another in the protein) but which are in close special proximity in the finally folded protein. It is well understood by those skilled in the art that epitopes require a minimum of six amino acid residues in order to be recognized by the immune system. Thus, in one embodiment the immunogenic portion from the influenza HA protein comprises at least one epitope. One embodiment of the present invention is a protein comprising at least 6 amino acids, at least 10 amino acids, at least 25 amino acids, at least 50 amino acids, at least 75 amino acids or at least 100 amino acids from the stem region of influenza HA protein. One embodiment of the present invention is a protein comprising at least 6 amino acids, at least 10 amino acids, at least 25 amino acids, at least 50 amino acids, at least 75 amino acids or at least 100 amino acids from the stem region of an HA protein from a virus selected from the group consisting of influenza type A viruses, influenza type B viruses and influenza type C viruses. One embodiment of the present invention is a protein comprising at least 6 amino acids, at least 10 amino acids, at least 25 amino acids, at least 50 amino acids, at least 75 amino acids or at least 100 amino acids from the stem region of an HA protein selected from the group consisting an H1 influenza virus HA protein, an H2 influenza virus HA protein, an influenza H3 virus HA protein, an influenza H4 virus HA protein, an influenza H5 virus HA protein, an influenza H6 virus HA protein, an H7 influenza virus HA protein, an H8 influenza virus HA protein, an H9 influenza virus HA protein, an H10 influenza virus HA protein HA protein, an H11 influenza virus HA protein, an H12 influenza virus HA protein, an H13 influenza virus HA protein, an H14 influenza virus HA protein, an H15 influenza virus HA protein and an H16 influenza virus HA protein. One embodiment of the present invention is a protein comprising at least 6 amino acids, at least 10 amino acids, at least 25 amino acids, at least 50 amino acids, at least 75 amino acids or at least 100 amino acids from the stem region of an HA protein from a strain of virus selected from the viruses listed in Table 2. In one embodiment, the amino acids are contiguous amino acids from the stem region of the HA protein. In one embodiment, such proteins comprising at least 6 amino acids, at least 10 amino acids, at least 25 amino acids, at least 50 amino acids, at least 75 amino acids or at least 100 amino acids from the stem region of an HA protein elicit the production of broadly neutralizing antibodies against influenza virus. One embodiment of the present invention is a protein comprising at least 6 amino acids, at least 10 amino acids, at least 25 amino acids, at least 50 amino acids, at least 75 amino acids or at least 100 amino acids from the stem region of an HA protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment, the amino acids are contiguous amino acids from the stem region of the HA protein. In one embodiment, the amino acids are non-contiguous, but are in close spatial proximity in the final protein.

While the present application discloses the use of stem regions from several exemplary HA proteins having specific sequences, the invention may also be practiced using stem regions from proteins comprising variations of the disclosed HA sequences. Thus, one embodiment of the present invention is a stem-region protein comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% identical the stem region of an HA protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. One embodiment of the present invention is a stem-region protein comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. One embodiment of the present invention is a stem-region protein comprising the stem region of an HA protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. One embodiment of the present invention is a stem-region protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98.

While the proteins disclosed thus far may elicit broadly neutralizing antibodies against an influenza virus, the inventors have discovered that such proteins are more stable and easier to purify when they exist in a trimeric form. Thus, one embodiment is a protein comprising the stem-region protein of the present invention joined to a trimerization domain. In one embodiment, the stem region is from an HA protein comprising an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment, the stem region is from an HA protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment, the stem region protein comprises an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one embodiment, the stem region protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one embodiment, the trimerization domain is selected from the group consisting of the HIV-1 gp41 trimerization domain, the SIV gp41 trimerization domain, the Ebola virus gp-2 trimerization domain, the HTLV-1 gp-21 trimerization domain, the T4 fibritin trimerization domain (i.e., foldon), the yeast heat shock transcription factor trimerization domain, and the human collagen trimerization domain. In one embodiment, the trimerization domain is an HIV gp41 trimerization domain.

The inventors have also found that, in some instances, stem region proteins of the present invention may be more stable when joined to at least part of the head region of the HA protein. Thus, one embodiment of the present invention is a protein comprising a stem region protein joined to the head region of an HA protein and a trimerization domain. In one embodiment, the stem region protein is from an HA protein comprising an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment, the stem region protein is from an HA protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment, the stem region protein comprises an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one embodiment, the stem region protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98.

In some embodiments of the present invention, the various protein domains (e.g., stem region protein, trimerization domain, head region, etc.) may be joined directly to one another. In other embodiments, it may be necessary to employ linkers (also referred to as a spacer sequences) so that the various domains are in the proper special orientation. The linker sequence is designed to position the hemagglutinin protein in such a way to that it maintains the ability to elicit an immune response to the influenza virus. Linker sequences of the present invention comprise amino acids. Preferable amino acids to use are those having small side chains and/or those which are not charged. Such amino acids are less likely to interfere with proper folding and activity of the fusion protein. Accordingly, preferred amino acids to use in linker sequences, either alone or in combination are serine, glycine and alanine Examples of such linker sequences include, but are not limited to, SGG, GSG, GG and NGTGGSG. Amino acids can be added or subtracted as needed. Those skilled in the art are capable of determining appropriate linker sequences for proteins of the present invention.

One embodiment of the present invention is a fusion protein comprising a stem region protein joined to at least a portion of the head region of an HA protein and a trimerization domain, wherein the fusion protein comprises one or more linker sequences. In one embodiment, the stem region protein is from an HA protein comprising an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment, the stem region protein is from an HA protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment, the stem region protein comprises an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one embodiment, the stem region protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one embodiment, the linker is selected from the group consisting of GG, GSG and NGTGGSG. In one embodiment, the protein elicits antibodies that neutralize at least one virus that is a different Type, sub-type or strain than the Type, sub-type or strain of the virus from which the HA protein was obtained.

Vaccines Comprising HA-Ferritin Fusion Proteins

The inventors have also discovered that fusion of influenza HA protein with ferritin protein (HA-ferritin fusion protein) results in a vaccine that elicits a robust immune response to influenza virus. Such HA-ferritin fusion proteins self-assemble into nanoparticles that display immunogenic portions of influenza hemagglutinin protein on their surface. These nanoparticles are useful for vaccinating individuals against a broad range of influenza viruses. Thus, one embodiment of the present invention is an HA-ferritin fusion protein comprising a monomeric ferritin subunit disclosed herein joined to an influenza hemagglutinin protein disclosed herein, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles.

Ferritin is a globular protein found in all animals, bacteria, and plants, that acts primarily to control the rate and location of polynuclear Fe(III)₂O₃ formation through the transportation of hydrated iron ions and protons to and from a mineralized core. The globular form of ferritin is made up of monomeric subunit proteins (also referred to as monomeric ferritin subunits), which are polypeptides having a molecule weight of approximately 17-20 kDa. An example of the sequence of one such monomeric ferritin subunit is represented by SEQ ID NO:2. Each monomeric ferritin subunit has the topology of a helix bundle which includes a four antiparallel helix motif, with a fifth shorter helix (the c-terminal helix) lying roughly perpendicular to the long axis of the 4 helix bundle. According to convention, the helices are labeled ‘A, B, C, and D & E’ from the N-terminus respectively. The N-terminal sequence lies adjacent to the capsid three-fold axis and extends to the surface, while the E helices pack together at the four-fold axis with the C-terminus extending into the particle core. The consequence of this packing creates two pores on the capsid surface. It is expected that one or both of these pores represent the point by which the hydrated iron diffuses into and out of the capsid. Following production, these monomeric ferritin subunit proteins self-assemble into the globular ferritin protein. Thus, the globular form of ferritin comprises 24 monomeric, ferritin subunit proteins, and has a capsid-like structure having 432 symmetry.

According to the present invention, a monomeric ferritin subunit of the present invention is a full length, single polypeptide of a ferritin protein, or any portion thereof, which is capable of directing self-assembly of monomeric ferritin subunits into the globular form of the protein. Amino acid sequences from monomeric ferritin subunits of any known ferritin protein can be used to produce fusion proteins of the present invention, so long as the monomeric ferritin subunit is capable of self-assembling into a nanoparticle displaying hemagglutinin on its surface. In one embodiment, the monomeric subunit is from a ferritin protein selected from the group consisting of a bacterial ferritin protein, a plant ferritin protein, an algal ferritin protein, an insect ferritin protein, a fungal ferritin protein and a mammalian ferritin protein. In one embodiment, the ferritin protein is from Helicobacter pylori.

HA-ferritin fusion proteins of the present invention need not comprise the full-length sequence of a monomeric subunit polypeptide of a ferritin protein. Portions, or regions, of the monomeric ferritin subunit protein can be utilized so long as the portion comprises an amino acid sequence that directs self-assembly of monomeric ferritin subunits into the globular form of the protein. One example of such a region is located between amino acids 5 and 167 of the Helicobacter pylori ferritin protein. More specific regions are described in Zhang, Y. Self-Assembly in the Ferritin Nano-Cage Protein Super Family. 2011, Int. J. Mol. Sci., 12, 5406-5421, which is incorporated herein by reference in its entirety.

One embodiment of the present invention is an HA-ferritin fusion protein comprising an HA protein of the present invention joined to at least 25 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, or at least 150 contiguous amino acids from a monomeric ferritin subunit, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles. One embodiment of the present invention is an HA-ferritin fusion protein comprising an HA protein of the present invention joined to at least 25 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, or at least 150 contiguous amino acids from the region of a ferritin protein corresponding to the amino acid sequences of the Helicobacter pylori ferritin monomeric subunit that direct self-assembly of the monomeric subunits into the globular form of the ferritin protein, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles. One embodiment of the present invention is an HA-ferritin fusion protein comprising an HA protein of the present invention joined to at least 25 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, or at least 150 contiguous amino acids from SEQ ID NO:2 that are capable of directing self-assembly of the monomeric subunits into the globular ferritin protein, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles. One embodiment of the present invention is an HA-ferritin fusion protein comprising an HA-protein of the present invention joined to at least 25 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, or at least 150 contiguous amino acids from amino acid residues 5-167 of SEQ ID NO:2, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles. One embodiment of the present invention is an HA-ferritin fusion protein comprising an HA protein of the present invention joined to at least 25 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, or at least 150 contiguous amino acids from SEQ ID NO:5, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles. One embodiment of the present invention is an HA-ferritin fusion protein comprising an HA protein of the present invention joined to amino acid residues 5-167 from SEQ ID NO:2, or SEQ ID NO:5, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles. As has been previously discussed, it is well-known in the art that some variations can be made in the amino acid sequence of a protein without affecting the activity of the protein. Such variations include insertion of amino acid residues, deletions of amino acid residues, and substitutions of amino acid residues. Thus, in one embodiment, the sequence of the monomeric ferritin subunit is divergent enough from the sequence of a ferritin subunit naturally found in a mammal, such that when the variant monomeric ferritin subunit is introduced into the mammal, it does not result in the production of antibodies that react with the mammal's natural ferritin protein. According to the present invention, such a monomeric subunit is referred to as immunogenically neutral. One embodiment of the present invention is an HA-ferritin fusion protein comprising an HA protein of the present invention joined to an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, and at least 97% identical to the amino acid sequence of a monomeric ferritin subunit that is responsible for directing self-assembly of the monomeric ferritin subunits into the globular form of the protein, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles. In one embodiment, the HA-ferritin fusion protein comprises a polypeptide sequence identical in sequence to a monomeric ferritin subunit. One embodiment of the present invention is an HA-ferritin fusion protein comprising an HA protein of the present invention joined to an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, and at least 97% identical to the amino acid sequence of a monomeric ferritin subunit from Helicobacter pylori, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles. One embodiment of the present invention is an HA-ferritin fusion protein comprising an HA protein of the present invention joined to an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, and at least 97% identical to a sequence selected from amino acid residues 5-167 from SEQ ID NO:2 and SEQ ID NO:5, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles. One embodiment of the present invention is an HA-ferritin fusion protein comprising an HA protein of the present invention joined to a sequence selected from amino acid residues 5-167 from SEQ ID NO:2 and SEQ ID NO:5.

In some embodiments, it may be useful to engineer mutations into the amino acid sequences of proteins of the present invention. For example, it may be useful to alter sites such as enzyme recognition sites or glycosylation sites in the monomeric ferritin subunit, the trimerization domain, or linker sequences, in order to give the fusion protein beneficial properties (e.g., solubility, half-life, mask portions of the protein from immune surveillance). In this regard, it is known that the monomeric subunit of ferritin is not glycosylated naturally. However, it can be glycosylated if it is expressed as a secreted protein in mammalian or yeast cells. Thus, in one embodiment, potential N-linked glycosylation sites in the amino acid sequences from the monomeric ferritin subunit are mutated so that the mutated ferritin subunit sequences are no longer glycosylated at the mutated site. One such sequence of a mutated monomeric ferritin subunit is represented by SEQ ID NO:5.

According to the present invention, the hemagglutinin protein portion of HA-ferritin fusion proteins of the present invention can be from any influenza virus, so long as the HA-ferritin fusion protein elicits an immune response against an influenza virus. Thus, one embodiment of the preset invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence from an HA protein from an influenza A virus, an influenza B virus or an influenza C virus. One embodiment of the preset invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence from an influenza A Group 1 virus HA protein. One embodiment of the preset invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence from an influenza A Group 2 virus HA protein. One embodiment of the preset invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence from an HA protein selected from the group consisting of an H1 influenza virus HA protein, an H2 influenza virus HA protein, an H5 influenza virus HA protein, an H7 virus influenza HA protein and an H9 influenza virus HA protein. One embodiment of the preset invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence from an HA protein selected from the group consisting of an H3 influenza virus HA protein, an H4 influenza virus HA protein, an H6 influenza virus HA protein, an H8 influenza virus HA protein, an H10 influenza virus HA protein, an H11 influenza virus HA protein, an H12 influenza virus HA protein, an H13 influenza virus HA protein, an H14 influenza virus HA protein, an H15 influenza virus HA protein, and an H15 influenza virus HA protein. One embodiment of the preset invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence of an HA protein from a virus listed in Table 2.

Preferred hemagglutinin proteins to use in constructing HA-ferritin fusion proteins of the present invention are those that elicit an immune response against an influenza virus. Even more preferred hemagglutinin proteins are those that are capable of eliciting antibodies to an influenza virus. One embodiment of the present invention is an HA-ferritin fusion protein that elicits antibodies to a virus selected from the group consisting of influenza A viruses, influenza B viruses and influenza C viruses. One embodiment of the present invention is a HA-ferritin fusion protein that elicits antibodies to a virus selected from the group consisting of an H1 influenza virus, an H2 influenza virus, an influenza H3 virus, an influenza H4 virus, an influenza H5 virus, an influenza H6 virus, an H7 influenza virus, an H8 influenza virus, an H9 influenza virus, an H10 influenza virus, an H11 influenza virus, an H12 influenza virus, an H13 influenza virus, an H14 influenza virus, an H15 influenza virus and an H16 influenza virus. One embodiment of the present invention is an HA-ferritin fusion protein that elicits antibodies to a virus listed in Table 2. Preferred antibodies elicited by HA-ferritin fusion proteins of the present invention are those that neutralize an influenza virus. Thus, one embodiment of the present invention is an HA-ferritin fusion protein that elicits neutralizing antibodies to a virus selected from the group consisting of influenza A viruses, influenza B viruses and influenza C viruses. One embodiment of the present invention is an HA-ferritin fusion protein that elicits neutralizing antibodies to a virus having a subtype selected from the group consisting of an H1 influenza virus, an H2 influenza virus, an influenza H3 virus, an influenza H4 virus, an influenza H5 virus, an influenza H6 virus, an H7 influenza virus, an H8 influenza virus, an H9 influenza virus, an H10 influenza virus, an H11 influenza virus, an H12 influenza virus, an H13 influenza virus, an H14 influenza virus, an H15 influenza virus and an H16 influenza virus. One embodiment of the present invention is an HA-ferritin fusion protein that elicits neutralizing antibodies to a virus listed in Table 2.

As has been discussed, neutralizing antibodies elicited by a HA-ferritin fusion protein of the present invention can neutralize viral infections by affecting any step in the life cycle of the virus. Thus, in one embodiment of the present invention, an HA-ferritin fusion protein elicits neutralizing antibodies that prevent influenza virus from attaching to the host cell. In one embodiment of the present invention, an HA-ferritin fusion protein may elicit neutralizing antibodies that prevent influenza virus from entering the host cell. In one embodiment of the present invention, an HA-ferritin fusion protein may elicit neutralizing antibodies that prevent fusion of viral membranes with endosomal membranes. In one embodiment of the present invention, an HA-ferritin fusion protein may elicit neutralizing antibodies that prevent influenza virus from releasing ribonucleoproteins into the cytoplasm of the host cell. In one embodiment of the present invention, an HA-ferritin fusion protein may elicit neutralizing antibodies that prevent assembly of new virus in the infected host cell. In one embodiment of the present invention, an HA-ferritin fusion protein may elicit neutralizing antibodies that prevent release of newly formed virus from the infected host cell.

Preferred HA-ferritin fusion proteins of the present invention are those that elicit broadly neutralizing antibodies. Thus, one embodiment is an HA-ferritin fusion protein that elicits antibodies that neutralizes more than one type of influenza virus selected from the group consisting of influenza type A viruses, influenza type B viruses and influenza type C viruses. One embodiment is an HA-ferritin fusion protein that elicits antibodies that neutralize more than one sub-type of influenza virus selected from the group consisting of an H1 influenza virus, an H2 influenza virus, an influenza H3 virus, an influenza H4 virus, an influenza H5 virus, an influenza H6 virus, an H7 influenza virus, an H8 influenza virus, an H9 influenza virus, an H10 influenza virus, an H11 influenza virus, an H12 influenza virus, an H13 influenza virus, an H14 influenza virus, an H15 influenza virus and an H16 influenza virus. One embodiment is an HA-ferritin protein that elicits antibodies that neutralize from more than one strain of influenza virus selected from the viruses listed in Table 2.

It will be understood by those skilled in the art that particularly useful HA-ferritin useful proteins of the present invention are those comprising an immunogenic portion of influenza HA protein. Thus, one embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to at least one immunogenic portion of an influenza HA protein. One embodiment of the present invention is an HA-ferritin protein comprising a ferritin protein of the present invention joined to at least one immunogenic portion of an HA protein from a virus selected from the group consisting of influenza type A viruses, influenza type B viruses and influenza type C viruses. One embodiment of the present invention is an HA-ferritin protein comprising a ferritin protein of the present invention joined to at least one immunogenic portion of an HA protein selected from the group consisting of an H1 influenza virus HA protein, an H2 influenza virus HA protein, an H5 influenza virus HA protein, an H7 virus influenza HA protein and an H9 influenza virus HA protein. One embodiment of the preset invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to at least one immunogenic portion of an HA protein selected from the group consisting of an H3 influenza virus HA protein, an H4 influenza virus HA protein, an H6 influenza virus HA protein, an H8 influenza virus HA protein, an H10 influenza virus HA protein, an H11 influenza virus HA protein, an H12 influenza virus HA protein, an H13 influenza virus HA protein, an H14 influenza virus HA protein, an H15 influenza virus HA protein, and an H16 influenza virus HA protein, joined to a ferritin protein of the present invention. One embodiment of the present invention is an HA-ferritin protein comprising a ferritin protein of the present invention joined to at least one immunogenic portion of an HA protein from virus listed in Table 2. In one embodiment, an HA-ferritin fusion protein comprising an immunogenic portion of an HA protein elicits the production of broadly neutralizing antibodies against influenza virus.

Immunogenic portions of proteins comprise epitopes, which are clusters of amino acid residues that are recognized by the immune system, thus eliciting an immune response. Such epitopes may consist of contiguous amino acids residues (i.e., amino acid residues that are adjacent to one another in the protein), or they may consist of non-contiguous amino acid residues (i.e., amino acid residues that are not adjacent one another in the protein) but which are in close special proximity in the finally folded protein. It is well understood by those skilled in the art that such epitopes require a minimum of six amino acid residues in order to be recognized by the immune system. Thus, one embodiment of the present invention is an HA-ferritin fusion comprising an immunogenic portion from the influenza HA protein, wherein the immunogenic portion comprises at least one epitope.

It is known in the art that some variation in a protein sequence can be tolerated without significantly affecting the activity of the protein. Thus, one embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence that is a variant of an HA protein from a virus selected from the group consisting of influenza Type A viruses influenza Type B viruses and influenza type C viruses. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the sequence of a HA protein from a virus selected from the group consisting of influenza Type A viruses influenza Type B viruses and influenza type C viruses, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the sequence of a HA protein selected from the group consisting an H1 influenza virus HA protein, an H2 influenza virus HA protein, an H5 influenza virus HA protein, an H7 virus influenza HA protein and an H9 influenza virus HA protein, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the sequence of a HA protein selected from the group consisting of an H3 influenza virus HA protein, an H4 influenza virus HA protein, an H6 influenza virus HA protein, an H8 influenza virus HA protein, an H10 influenza virus HA protein, an H11 influenza virus HA protein, an H12 influenza virus HA protein, an H13 influenza virus HA protein, an H14 influenza virus HA protein, an H15 influenza virus HA protein, and an H16 influenza virus HA protein, joined to a ferritin protein of the present invention, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the sequence of a HA protein from a virus listed in Table 2, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38.

One embodiment of the present invention is an HA-ferritin fusion protein comprising an amino acid sequence at least 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68. One embodiment of the present invention is an HA-ferritin fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68.

It is known in the art that influenza hemagglutinin proteins have various regions, or domains, each possessing specific activities. For example, the globular head extends out from the lipid membrane and comprises two domains: the receptor binding domain (RBD) and the vestigial esterase domain. The RB domain is involved in binding of the HA protein to receptors. The globular head also includes several antigenic sites that include immunodominant epitopes. The stem region is responsible for anchoring the HA protein into the viral lipid envelope. Thus, it will be understood by those skilled in the art that HA-ferritin fusion proteins of the present invention need not comprise the entire sequence of the HA protein. Instead, an HA-ferritin fusion protein can comprise only those portions, regions, domains, and the like, that contain the necessary activities for practicing the present invention. For example, an HA-ferritin fusion protein may contain only those amino acid sequences from the HA protein that contain antigenic sites, epitopes, immunodominant epitopes, and the like.

One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, or at least 500 amino acids from an HA protein from a virus selected from the group consisting of influenza Type A viruses influenza Type B viruses and influenza type C viruses, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, or at least 500 amino acids from an HA protein selected from the group consisting an H1 influenza virus HA protein, an H2 influenza virus HA protein, an H5 influenza virus HA protein, an H7 virus influenza HA protein and an H9 influenza virus HA protein, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, or at least 500 amino acids from an HA protein selected from the group consisting of an H3 influenza virus HA protein, an H4 influenza virus HA protein, an H6 influenza virus HA protein, an H8 influenza virus HA protein, an H10 influenza virus HA protein, an H11 influenza virus HA protein, an H12 influenza virus HA protein, an H13 influenza virus HA protein, an H14 influenza virus HA protein, an H15 influenza virus HA protein, and an H16 influenza virus HA protein, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, or at least 500 amino acids from and HA protein from a virus listed in Table 2, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against in influenza virus. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, or at least 500 amino acids from a protein consisting of a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38.

One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to at least one domain from a HA protein from a virus listed in Table 2, wherein the domain is selected from the group consisting of an ectodomain, an RDB domain, a stem domain, and a domain comprising the region stretching from the amino acid residue immediately distal to the last amino acid of second helical coil to the amino acid residue proximal to the first amino acid of the transmembrane domain. According to the present invention, an ectodomain of an influenza hemagglutinin protein refers to the portion of the hemagglutinin protein that lies outside its transmembrane domain. In one embodiment, the HA-ferritin fusion protein comprises a ferritin protein of the present invention joined to a region of a HA protein from a virus listed in Table 2, wherein the region consists of the amino acid immediately distal to the last amino acid of the second helical coiled coil and proximal to the first amino acid of the transmembrane domain. In one embodiment, the HA-ferritin fusion protein comprises a ferritin protein of the present invention joined to a region of a HA protein from a virus listed in Table 2, wherein the region comprises an amino acid sequence distal to the second helical coiled coil and proximal to the transmembrane domain. In one embodiment, the HA-ferritin fusion protein comprises a ferritin protein of the present invention joined to the ectodomain of a HA protein from a virus listed in Table 2. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38.

The stem region of an influenza HA protein is a particularly useful domain for constructing fusion proteins of the present invention. Thus, one embodiment of the present invention is a ferritin protein of the present invention joined to at least one immunogenic portion from the stem region of influenza HA protein. According to the preset invention, such a protein is referred to an HA SS-ferritin fusion protein. As used herein, the HA stem region of the hemagglutinin protein consists of the amino acids from the membrane up to the head region of the protein. More specifically, the stem region consists of the amino terminal amino acid up to the cysteine at position 52, and all residues after the cysteine residue at position 277 (using standard H3 numbering). Sequences of exemplary stem regions are represented by SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98.

One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, or at least 200 amino acids from the stem region of an HA protein from a virus selected from the group consisting of influenza Type A viruses influenza Type B viruses and influenza type C viruses, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, or at least 200 amino acids from the stem region of an HA protein selected from the group consisting an H1 influenza virus HA protein, an H2 influenza virus HA protein, an H5 influenza virus HA protein, an H7 virus influenza HA protein and an H9 influenza virus HA protein, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, or at least 200 amino acids from the stem region of an HA protein selected from the group consisting of an H3 influenza virus HA protein, an H4 influenza virus HA protein, an H6 influenza virus HA protein, an H8 influenza virus HA protein, an H10 influenza virus HA protein, an H11 influenza virus HA protein, an H12 influenza virus HA protein, an H13 influenza virus HA protein, an H14 influenza virus HA protein, an H15 influenza virus HA protein, and an H16 influenza virus HA protein, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, or at least 200 amino acids from the stem region of an HA protein from a virus listed in Table 2, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against in influenza virus. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, or at least 200 amino acids from the stem region of an HA protein comprising a sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, or at least 200 amino acids from the stem region comprising a sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98.

One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the sequence of the stem region of an HA protein from a virus selected from the group consisting of influenza Type A viruses influenza Type B viruses and influenza type C viruses, wherein the Ha-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the sequence of the stem region of an HA protein selected from the group consisting an H1 influenza virus HA protein, an H2 influenza virus HA protein, an H5 influenza virus HA protein, an H7 virus influenza HA protein and an H9 influenza virus HA protein, wherein the Ha-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the sequence of the stem region of an HA protein selected from the group consisting of an H3 influenza virus HA protein, an H4 influenza virus HA protein, an H6 influenza virus HA protein, an H8 influenza virus HA protein, an H10 influenza virus HA protein, an H11 influenza virus HA protein, an H12 influenza virus HA protein, an H13 influenza virus HA protein, an H14 influenza virus HA protein, an H15 influenza virus HA protein, and an H16 influenza virus HA protein, joined to a ferritin protein of the present invention, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the sequence of the stem region of an HA protein from a virus listed in Table 2, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the stem region of an HA protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38., wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98.

As has been described for stem region proteins of the present invention, the inventors have found that HA-ferritin fusion proteins are more stable and easier to purify when they exist in a trimeric form. Thus, in one embodiment of the present invention the HA portion of the HA-ferritin fusion protein is joined to one or more trimerization domains. In one embodiment, the HA protein comprises an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38, joined to one or more trimerization domains. In one embodiment, the HA protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38 joined to one or more trimerization domains. In one embodiment, the HA protein comprises an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98 joined to one or more trimerization domains. In one embodiment, the HA protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98 joined to one or more trimerization domains. In one embodiment, the trimerization domain is selected from the group consisting of the HIV-1 gp41 trimerization domain, the SIV gp41 trimerization domain, the Ebola virus gp-2 trimerization domain, the HTLV-1 gp-21 trimerization domain, the T4 fibritin trimerization domain (i.e., foldon), the yeast heat shock transcription factor trimerization domain, and the human collagen trimerization domain. In one embodiment, the trimerization domain is an HIV gp41 trimerization domain.

Additionally, the inventors have found that, in some instances, HA-ferritin fusion proteins in which the HA portion is limited to HA stem region sequences may be more stable when joined to at least part of the head region of the HA protein. Thus, one embodiment of the present invention is an HA 55-ferritin fusion protein, wherein, the HA portion of the fusion protein is joined to an amino acid sequence from at least a portion of an HA protein head region.

HA-ferritin proteins of the present invention are constructed by joining ferritin proteins of the present invention with HA proteins of the present invention. In addition, HA-ferritin fusion proteins may contain other domains (e.g., stem region protein, trimerization domain, head region, etc.) that improve the functionality of the final HA-ferritin fusion protein. In some embodiments, joining of the various proteins and/or domains can be done such that the sequences are directly linked. In other embodiments, it may be necessary to employ linkers (also referred to as a spacer sequences) between the various proteins and/or domains so that the so that they are in the proper special orientation. More specifically, linker sequence can be inserted so that the hemagglutinin protein is positioned in such a way to maintain the ability to elicit an immune response to the influenza virus. Linker sequences of the present invention comprise amino acids. Preferable amino acids to use are those having small side chains and/or those which are not charged. Such amino acids are less likely to interfere with proper folding and activity of the fusion protein. Accordingly, preferred amino acids to use in linker sequences, either alone or in combination are serine, glycine and alanine Examples of such linker sequences include, but are not limited to, SGG, GSG, GG and NGTGGSG. Amino acids can be added or subtracted as needed. Those skilled in the art are capable of determining appropriate linker sequences for proteins of the present invention.

In accordance with the invention, suitable portions of the hemagglutinin protein can be joined to the ferritin protein either as an exocapsid product by fusion with the N-terminal sequence lying adjacent to the capsid three-fold axis, as an endocapsid product by fusion with the C-terminus extending inside the capsid core, or a combination thereof. In one embodiment, the hemagglutinin portion of the fusion protein is joined to the N-terminal sequence of the ferritin portion of the fusion protein.

One embodiment of the present invention is an HA-ferritin fusion protein comprising an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128, wherein the HA-ferritin fusion protein elicits the production of neutralizing antibodies against an influenza protein. One embodiment of the present invention is an HA-ferritin fusion protein comprising SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128.

Proteins of the present invention are encoded by nucleic acid molecules of the present invention. In addition, they are expressed by nucleic acid constructs of the present invention. As used herein a nucleic acid construct is a recombinant expression vector, i.e., a vector linked to a nucleic acid molecule encoding a protein such that the nucleic acid molecule can effect expression of the protein when the nucleic acid construct is administered to, for example, a subject or an organ, tissue or cell. The vector also enables transport of the nucleic acid molecule to a cell within an environment, such as, but not limited to, an organism, tissue, or cell culture. A nucleic acid construct of the present disclosure is produced by human intervention. The nucleic acid construct can be DNA, RNA or variants thereof. The vector can be a DNA plasmid, a viral vector, or other vector. In one embodiment, a vector can be a cytomegalovirus (CMV), retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poliovirus, sindbis virus, or any other DNA or RNA virus vector. In one embodiment, a vector can be a pseudotyped lentiviral or retroviral vector. In one embodiment, a vector can be a DNA plasmid. In one embodiment, a vector can be a DNA plasmid comprising viral components and plasmid components to enable nucleic acid molecule delivery and expression. Methods for the construction of nucleic acid constructs of the present disclosure are well known. See, for example, Molecular Cloning: a Laboratory Manual, 3^(rd) edition, Sambrook et al. 2001 Cold Spring Harbor Laboratory Press, and Current Protocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons, 1994. In one embodiment, the vector is a DNA plasmid, such as a CMV/R plasmid such as CMV/R or CMV/R 8 KB (also referred to herein as CMV/R 8 kb). Examples of CMV/R and CMV/R 8 kb are provided herein. CMV/R is also described in U.S. Pat. No. 7,094,598 B2, issued Aug. 22, 2006.

As used herein, a nucleic acid molecule comprises a nucleic acid sequence that encodes a stem region immunogen, a ferritin monomeric subunit, a hemagglutinin protein, and/or an HA-ferritin fusion protein of the present invention. A nucleic acid molecule can be produced recombinantly, synthetically, or by a combination of recombinant and synthetic procedures. A nucleic acid molecule of the disclosure can have a wild-type nucleic acid sequence or a codon-modified nucleic acid sequence to, for example, incorporate codons better recognized by the human translation system. In one embodiment, a nucleic acid molecule can be genetically-engineered to introduce, or eliminate, codons encoding different amino acids, such as to introduce codons that encode an N-linked glycosylation site. Methods to produce nucleic acid molecules of the disclosure are known in the art, particularly once the nucleic acid sequence is know. It is to be appreciated that a nucleic acid construct can comprise one nucleic acid molecule or more than one nucleic acid molecule. It is also to be appreciated that a nucleic acid molecule can encode one protein or more than one protein.

Preferred nucleic acid molecules are those that encode a stem-region protein, a ferritin monomeric subunit, a hemagglutinin protein, and/or an HA-ferritin fusion protein comprising a monomeric subunit of a ferritin protein joined to an influenza hemagglutinin protein. Thus, one embodiment of the present invention is a nucleic acid molecule comprising a nucleic acid sequence encoding a protein that comprises a monomeric subunit of a ferritin protein joined to an influenza hemagglutinin protein. In one embodiment, the monomeric subunit of ferritin is from the ferritin protein of Helicobacter pylori. In one embodiment, the monomeric subunit comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5. In one embodiment, the monomeric subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5. In one embodiment the influenza hemagglutinin protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment the influenza hemagglutinin protein comprises a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment the influenza hemagglutinin protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one embodiment the influenza hemagglutinin protein comprises a sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one embodiment the influenza hemagglutinin protein comprises at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, or at least 200 amino acids from a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment the influenza hemagglutinin protein comprises at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, or at least 200 amino acids from a sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98.

One embodiment of the present invention is a nucleic acid molecule comprising a nucleic sequence encoding a protein comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68. One embodiment of the present invention is a nucleic acid molecule comprising a nucleic sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68.

One embodiment of the present invention is a nucleic acid molecule comprising a nucleic sequence encoding a protein comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% identical to SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128. One embodiment of the present invention is a nucleic acid molecule comprising a nucleic sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128.

Also embodied in the present invention are nucleic acid sequences that are variants of nucleic acid sequence encoding protein of the present invention. Such variants include nucleotide insertions, deletions, and substitutions, so long as they do not affect the ability of fusion proteins of the present invention to self-assemble into nanoparticles, or significantly affect the ability of the hemagglutinin portion of fusion proteins to elicit an immune response to an influenza virus. Thus, one embodiment of the present invention is a nucleic acid molecule encoding a fusion protein of the present invention, wherein the monomeric subunit is encoded by a nucleotide sequence at least 85%, at least 90%, at least 95%, or at least 97% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4. One embodiment of the present invention is a nucleic acid molecule encoding an HA-ferritin fusion protein of the present invention, wherein the HA protein is encoded by a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 97% identical or at least 99% identical to a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. One embodiment of the present invention is a nucleic acid molecule encoding an HA-ferritin fusion protein of the present invention, wherein the HA protein is encoded by a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 97% identical or at least 99% identical to a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98.

One embodiment of the present invention is a nucleic acid molecule comprising a nucleic acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, and SEQ ID NO:37. One embodiment of the present invention is a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, and SEQ ID NO:37.

One embodiment of the present invention is a nucleic acid molecule comprising a nucleic acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, and SEQ ID NO:67. One embodiment of the present invention is a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, and SEQ ID NO:67.

One embodiment of the present invention is a nucleic acid molecule comprising a nucleic acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:94, and SEQ ID NO:97. One embodiment of the present invention is a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:94, and SEQ ID NO:97.

One embodiment of the present invention is a nucleic acid molecule comprising a nucleic acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:121, SEQ ID NO:124, and SEQ ID NO:127. One embodiment of the present invention is a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:121, SEQ ID NO:124, and SEQ ID NO:127.

Also encompassed by the present invention are expression systems for producing fusion proteins of the present invention. In one embodiment, nucleic acid molecules of the present invention are operationally linked to a promoter. As used herein, operationally linked means that proteins encoded by the linked nucleic acid molecules can be expressed when the linked promoter is activated. Promoters useful for practicing the present invention are known to those skilled in the art. One embodiment of the present invention is a recombinant cell comprising a nucleic acid molecule of the present invention. One embodiment of the present invention is a recombinant virus comprising a nucleic acid molecule of the present invention.

As indicated above, the recombinant production of the ferritin fusion proteins of the present invention can take place using any suitable conventional recombinant technology currently known in the field. For example, molecular cloning a fusion protein, such as ferritin with a suitable protein such as the recombinant influenza hemagglutinin protein, can be carried out via expression in E. coli with the suitable monomeric subunit protein, such as the helicobacter pylori ferritin monomeric subunit. The construct may then be transformed into protein expression cells, grown to suitable size, and induced to produce the fusion protein.

As has been described, because HA-ferritin fusion proteins of the present invention comprise a monomeric subunit of ferritin, they can self-assemble. According to the present invention, the supramolecule resulting from such self-assembly is referred to as a hemagglutinin expressing ferritin based nanoparticle. For ease of discussion, the hemagglutinin expressing ferritin based nanoparticle will simply be referred to as a, or the, nanoparticle (np). Nanoparticles of the present invention have the same structural characteristics as the ferritin proteins described earlier. That is, they contain 24 subunits and have 432 symmetry. In the case of nanoparticles of the present invention, the subunits are the fusion proteins comprising a ferritin monomeric subunit joined to an influenza hemagglutinin protein. Such nanoparticles display at least a portion of the hemagglutinin protein on their surface as hemagglutinin trimers. In such a construction, the hemagglutinin trimer is accessible to the immune system and thus can elicit an immune response. Thus, one embodiment of the present invention is a nanoparticle comprising an HA-ferritin fusion protein, wherein the fusion protein comprises a monomeric ferritin subunit joined to an influenza hemagglutinin protein. In one embodiment, the nanoparticle is an octahedron. In one embodiment, the nanoparticle displays the hemagglutinin protein on its surface as a hemagglutinin trimer. In one embodiment, the influenza hemagglutinin protein is capable of eliciting neutralizing antibodies to an influenza virus. In one embodiment, the monomeric ferritin subunit comprises at least 50 amino acids, at least 100 amino acids, or at least 150 amino acids from an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5, and/or comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 97% at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5. In one embodiment, the monomeric ferritin subunit comprises SEQ ID NO:2 or SEQ ID NO:5.

In one embodiment, the influenza hemagglutinin protein comprises at least one epitope from an influenza hemagglutinin protein listed in Table 2. In one embodiment, the influenza hemagglutinin protein comprises at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, or at least 500 amino acids from a hemagglutinin protein of a virus listed in Table 2. In one embodiment, the hemagglutinin protein comprises at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, or at least 500 amino acids from a protein consisting of a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment, the hemagglutinin protein comprises a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38.

In one embodiment, the influenza hemagglutinin protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to the sequence of an hemagglutinin protein from a virus listed in Table 2. In one embodiment, the influenza hemagglutinin protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to a protein sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38.

In one embodiment, the hemagglutinin protein comprises at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, or at least 500 amino acids from a protein consisting of a sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one embodiment, the influenza hemagglutinin protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to a protein sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one embodiment, the hemagglutinin protein comprises a sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98.

In one embodiment, the HA-ferritin fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to a protein sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68. In one embodiment, the HA-ferritin fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68. In one embodiment, the HA-ferritin fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128. In one embodiment, the HA-ferritin fusion protein comprises SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128.

Because stem region immunogens, HA-ferritin fusion proteins and nanoparticles of the present invention can elicit an immune response to an influenza virus, they can be used as vaccines to protect individuals against infection by influenza virus. According to the present invention a vaccine can be a stem region immunogen, an HA-ferritin fusion protein, or a nanoparticle of the present invention. Thus, one embodiment of the present invention is a vaccine comprising a stem region immunogen, an HA-ferritin fusion protein, or a nanoparticle of the present invention. Vaccines of the present invention can also contain other components such as adjuvants, buffers and the like. Although any adjuvant can be used, preferred embodiments can contain: chemical adjuvants such as aluminum phosphate, benzyalkonium chloride, ubenimex, and QS21; genetic adjuvants such as the IL-2 gene or fragments thereof, the granulocyte macrophage colony-stimulating factor (GM-CSF) gene or fragments thereof, the IL-18 gene or fragments thereof, the chemokine (C—C motif) ligand 21 (CCL21) gene or fragments thereof, the IL-6 gene or fragments thereof, CpG, LPS, TLR agonists, and other immune stimulatory genes; protein adjuvants such IL-2 or fragments thereof, the granulocyte macrophage colony-stimulating factor (GM-CSF) or fragments thereof, IL-18 or fragments thereof, the chemokine (C—C motif) ligand 21 (CCL21) or fragments thereof, IL-6 or fragments thereof, CpG, LPS, TLR agonists and other immune stimulatory cytokines or fragments thereof; lipid adjuvants such as cationic liposomes, N3 (cationic lipid), monophosphoryl lipid A (MPL1); other adjuvants including cholera toxin, enterotoxin, Fms-like tyrosine kinase-3 ligand (Flt-3L), bupivacaine, marcaine, and levamisole.

One embodiment of the disclosure is a ferritin-based nanoparticle vaccine that includes more than one influenza hemagglutinin protein. Such a vaccine can include a combination of different influenza hemagglutinin proteins, either on a single nanoparticle or as a mixture of nanoparticles, at least two of which have a unique influenza hemagglutinin proteins. A multivalent vaccine can comprise as many influenza hemagglutinin proteins as necessary in order to result in production of the immune response necessary to protect against a desired breadth of virus strains. In one embodiment, the vaccine comprises a hemagglutinin protein from at least two different influenza strains (bi-valent). In one embodiment, the vaccine comprises a hemagglutinin protein from at least three different influenza strains (tri-valent). In one embodiment, the vaccine comprises a hemagglutinin protein from at least four different influenza strains (tetra-valent). In one embodiment, the vaccine comprises a hemagglutinin protein from at least five different influenza strains (penta-valent). In one embodiment, the vaccine comprises a hemagglutinin protein from at least six different influenza strains (hexa-valent). In various embodiments, a vaccine comprises a hemagglutinin protein from each of 7, 8, 9, or 10 different strains of influenza virus. An example of such a combination is a ferritin-based nanoparticle vaccine that comprises influenza A group 1 hemagglutinin protein, an influenza A group 2 hemagglutinin protein, and an influenza B hemagglutinin protein. In one embodiment, the influenza hemagglutinin proteins are H1 HA, H3 HA, and B HA. In one embodiment, the influenza hemagglutinin proteins are those included in the 2011-2012 influenza vaccine. Another example of a multivalent vaccine is a ferritin based nanoparticle vaccine that comprises hemagglutinin proteins from four different influenza viruses. In one embodiment, the multivalent vaccine comprises hemagglutinin proteins from H1 A/NC/20/1999, H1 A/CA/04/2009, H2 A/Singapore/1/1957 and H5 A/Indonesia/05/2005. Such a vaccine is described in Example 2.

One embodiment of the present invention is a method to vaccinate an individual against influenza virus, the method comprising administering a nanoparticle to an individual such that an immune response against influenza virus is produced in the individual, wherein the nanoparticle comprises a monomeric subunit from ferritin joined to an influenza hemagglutinin protein, and wherein the nanoparticle displays the influenza hemagglutinin on its surface. In one embodiment, the nanoparticle is a monovalent nanoparticle. In one embodiment, the nanoparticle is multivalent nanoparticle. Another embodiment of the present invention is a method to vaccinate an individual against infection with influenza virus, the method comprising:

a) obtaining a nanoparticle comprising monomeric subunits, wherein the monomeric subunits comprise a ferritin protein joined to an influenza hemagglutinin protein, and wherein the nanoparticle displays the influenza hemagglutinin on its surface; and,

b) administering the nanoparticle to an individual such that an immune response against an influenza virus is produced.

One embodiment of the present invention is a method to vaccinate an individual against influenza virus, the method comprising administering a vaccine of the embodiments to an individual such that an immune response against influenza virus is produced in the individual, wherein the vaccine comprises at least one nanoparticle comprising a monomeric subunit from ferritin joined to an influenza hemagglutinin protein, and wherein the nanoparticle displays the influenza hemagglutinin on its surface. In one embodiment, the vaccine is a stem region immunogen. In one embodiment, the vaccine is a nanoparticle. In one embodiment, the vaccine is a monovalent vaccine. In one embodiment, the vaccine is multivalent vaccine. Another embodiment of the present invention is a method to vaccinate an individual against infection with influenza virus, the method comprising:

a) obtaining a vaccine comprising at least one nanoparticle comprising an HA-ferritin fusion protein, wherein the fusion protein comprises a ferritin protein joined to an influenza HA protein, and wherein the nanoparticle displays the influenza HA on its surface; and,

b) administering the vaccine to an individual such that an immune response against an influenza virus is produced.

In one embodiment, the nanoparticle is a monovalent nanoparticle. In one embodiment, the nanoparticle is multivalent nanoparticle.

In one embodiment, the nanoparticle is an octahedron. In one embodiment, the influenza hemagglutinin protein is capable of eliciting neutralizing antibodies to an influenza virus. In one embodiment, the influenza HA protein is capable of eliciting broadly neutralizing antibodies to an influenza virus. In one embodiment, the ferritin portion of the fusion protein comprise at least 50 amino acids, at least 100 amino acids, or at least 150 amino acids from an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5, and/or comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 97% at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5. In one embodiment, the HA portion of the fusion protein comprises at least one epitope from an influenza hemagglutinin protein listed in Table 2. In one embodiment, the HA portion of the fusion protein comprises at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, or at least 500 amino acids from a hemagglutinin protein of a virus listed in Table 2. In one embodiment, the HA portion of the fusion protein comprises at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, or at least 500 amino acids from a protein consisting of a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment, the HA portion of the fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to the sequence of an HA protein from a virus listed in Table 2. In one embodiment, the HA portion of the fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment, the HA portion of the fusion protein comprises at least 25 amino acids, at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, or at least 500 amino acids from a sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one embodiment, the HA portion of the fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one embodiment, the HA-ferritin fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to a protein sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68. In one embodiment, the HA-ferritin fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68. In one embodiment, the HA-ferritin fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128. In one embodiment, the HA-ferritin fusion protein comprises SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128.

Vaccines of the present invention can be used to vaccinate individuals using a prime/boost protocol. Such a protocol is described in U.S. Patent Publication No. 20110177122, which is incorporated herein by reference in its entirety. In such a protocol, a first vaccine composition may be administered to the individual (prime) and then after a period of time, a second vaccine composition may be administered to the individual (boost). Administration of the boosting composition is generally weeks or months after administration of the priming composition, preferably about 2-3 weeks or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks. In one embodiment, the boosting composition is formulated for administration about 1 week, or 2 weeks, or 3 weeks, or 4 weeks, or 5 weeks, or 6 weeks, or 7 weeks, or 8 weeks, or 9 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks after administration of the priming composition.

The first and second vaccine compositions can be, but need not be, the same composition. Thus, in one embodiment of the present invention, the step of administering the vaccine comprises administering a first vaccine composition, and then at a later time, administering a second vaccine composition. In one embodiment, the first vaccine composition comprises a nanoparticle comprising an HA-ferritin fusion protein of the present invention. In one embodiment, the first vaccine composition comprises a nanoparticle comprising an ectodomain from the hemagglutinin protein of an influenza virus selected from the group consisting of A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009 CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968 (1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo, B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris, H1), B/Brisbane/60/2008 (2008 Bris, B). In one embodiment, the hemagglutinin of the first vaccine composition comprises an amino acid sequence at least about 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment, the first vaccine composition comprises an HA-ferritin fusion protein comprising an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68, wherein the nanoparticle elicits an immune response against an influenza virus. In one embodiment, the first vaccine composition comprises an HA-ferritin fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68. In one embodiment, the second vaccine composition comprises a nanoparticle comprising an HA SS-ferritin fusion protein of the present invention. In one embodiment, the HA SS-ferritin fusion protein comprises an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98. In one embodiment, the HA SS-ferritin fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98. In one embodiment, the HA 55-ferritin fusion protein comprises an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical or at least 99% identical to SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128, wherein the HA 55-ferritin fusion protein elicits an immune response to an influenza virus. In one embodiment, the HA SS-ferritin fusion protein comprises SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128. In one embodiment, the individual is at risk for infection with influenza virus. In one embodiment, the individual has been exposed to influenza virus. As used herein, the terms exposed, exposure, and the like, indicate the subject has come in contact with a person of animal that is known to be infected with an influenza virus. Vaccines of the present invention may be administered using techniques well known to those in the art. Techniques for formulation and administration may be found, for example, in “Remington's Pharmaceutical Sciences”, 18^(th) ed., 1990, Mack Publishing Co., Easton, Pa. Vaccines may be administered by means including, but not limited to, traditional syringes, needleless injection devices, or microprojectile bombardment gene guns. Suitable routes of administration include, but are not limited to, parenteral delivery, such as intramuscular, intradermal, subcutaneous, intramedullary injections, as well as, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few. For injection, the compounds of one embodiment of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.

In one embodiment, vaccines, or nanoparticles, of the present invention can be used to protect an individual against infection by heterologous influenza virus. That is, a vaccine made using hemagglutinin protein from one strain of influenza virus is capable of protecting an individual against infection by different strains of influenza. For example, a vaccine made using hemagglutinin protein from influenza A/New Calcdonia/20/1999 (1999 NC, H1), can be used to protect an individual against infection by an influenza virus including, but not limited to A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009 CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968 (1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005 (2005 indo, H5), A/Perth/16/2009 (2009 Per, H3), and/or A/Brisbane/59/2007 (2007 Bris, H1).

In one embodiment, vaccines, or nanoparticles, of the present invention can be used to protect an individual against infection by an antigenically divergent influenza virus. Antigenically divergent refers to the tendency of a strain of influenza virus to mutate over time, thereby changing the amino acids that are displayed to the immune system. Such mutation over time is also referred to as antigenic drift. Thus, for example, a vaccine made using hemagglutinin protein from a A/New Calcdonia/20/1999 (1999 NC, H1) strain of influenza virus is capable of protecting an individual against infection by earlier, antigenically divergent New Calcdonia strains of influenza, and by evolving (or diverging) influenza strains of the future.

Because nanoparticles of the present invention display hemagglutinin proteins that are antigenically similar to an intact hemagglutinin, they can be used in assays for detecting antibodies against influenza virus (anti-influenza antibodies).

Thus, one embodiment of the present invention is a method for detecting anti-influenza virus antibodies using nanoparticles of the present invention. A detection method of the present invention can generally be accomplished by:

a. contacting at least a portion of a sample being tested for the presence of anti-influenza antibodies with a nanoparticle of the present invention; and,

b. detecting the presence of a nanoparticle/antibody complex;

wherein the presence of a nanoparticle/antibody complex indicates that the sample contains anti-influenza antibodies.

In one embodiment of the present invention, a sample is obtained, or collected, from an individual to be tested for the presence of anti-influenza virus antibodies. The individual may or may not be suspected of having anti-influenza antibodies or of having been exposed to influenza virus. A sample is any specimen obtained from the individual that can be used to test for the presence of anti-influenza virus antibodies. A preferred sample is a body fluid that can be used to detect the presence of anti-influenza virus antibodies. Examples of body fluids that may be used to practice the present method include, but are not limited to, blood, plasma, serum, lacrimal fluid and saliva. Those skilled in the art can readily identify samples appropriate for practicing the disclosed methods.

Blood, or blood-derived fluids such as plasma, serum, and the like, are particularly suitable as the sample. Such samples can be collected and prepared from individuals using methods known in the art. The sample may be refrigerated or frozen before assay.

Any nanoparticle of the present invention can be used to practice the disclosed method as long as the nanoparticle binds to anti-influenza virus antibodies. Useful nanoparticles, and methods of their production, have been described in detail herein. In a preferred embodiment, the nanoparticle comprises a fusion protein, wherein the fusion protein comprises at least 25, at least 50, at least 75, at least 100, or at least 150 contiguous amino acids from a monomeric ferritin subunit protein joined to (fused to) at least one epitope from an influenza hemagglutinin protein (i.e., an HA-ferritin fusion protein) such that the nanoparticle comprises trimers of the influenza virus HA protein epitope on its surface, and wherein the fusion protein is capable of self-assembling into nanoparticles. In one embodiment the at least 25, at least 50, at least 75, at least 100, or at least 150 contiguous amino acids are from the region of a monomeric ferritin protein corresponding to the amino acid sequences of the Helicobacter pylori ferritin monomeric subunit that direct self-assembly of the monomeric subunits into the globular form of the ferritin protein. In one embodiment the at least 25, at least 50, at least 75, at least 100, or at least 150 contiguous amino acids are from SEQ ID NO:2, and are capable of directing self-assembly of the monomeric subunits into the globular ferritin protein. In one embodiment the at least 25, at least 50, at least 75, at least 100, or at least 150 contiguous amino acids are from amino acid residues 5-167 of SEQ ID NO:2, or from SEQ ID NO:5, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles.

In one embodiment the nanoparticle comprises a fusion protein comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identical to the amino acid sequence of a monomeric ferritin subunit that is responsible for directing self-assembly of the monomeric ferritin subunits into the globular form of the protein, wherein the fusion protein is capable of self-assembling into nanoparticles. In one embodiment, the fusion protein comprises a polypeptide sequence identical in sequence to a monomeric ferritin subunit. In one embodiment the fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identical to the amino acid sequence of a monomeric ferritin subunit from Helicobacter pylori, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles. In one embodiment the fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identical to the amino acid sequence of to a sequence selected from the group consisting of (1) amino acid residues 5-167 from SEQ ID NO:2 and (2) SEQ ID NO:5, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles. In one embodiment the fusion protein comprises a sequence selected from the group consisting of (1) amino acid residues 5-167 from SEQ ID NO:2 and (2) SEQ ID NO:5.

In one embodiment, the nanoparticle comprises a fusion protein comprising a ferritin protein of the present invention joined to at least one immunogenic portion of an HA protein from a virus selected from the group consisting of influenza type A viruses, influenza type B viruses and influenza type C viruses. In one embodiment the fusion protein comprises a ferritin protein of the present invention joined to at least one immunogenic portion of an HA protein selected from the group consisting of an H1 influenza virus HA protein, an H2 influenza virus HA protein, H3 influenza virus HA protein, an H4 influenza virus HA protein, an H5 influenza virus HA protein, an H6 influenza virus HA protein, an H7 virus influenza HA protein, an H8 influenza virus HA protein, an H9 influenza virus HA protein, an H10 influenza virus HA protein, an H11 influenza virus HA protein, an H12 influenza virus HA protein, an H13 influenza virus HA protein, an H14 influenza virus HA protein, an H15 influenza virus HA protein, and an H16 influenza virus HA protein. In one embodiment, the fusion protein comprises a ferritin protein of the present invention joined to at least one immunogenic portion of an HA protein from virus listed in Table 2. In, one embodiment the immunogenic portion comprises at least one epitope.

In one embodiment, the nanoparticle comprises a fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence that is a variant of an HA protein from a virus selected from the group consisting of influenza Type A viruses influenza type B viruses and influenza type C viruses, wherein the fusion protein is capable of selectively binding anti-influenza antibodies. In one embodiment, the fusion protein comprises a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the sequence of a HA protein from a virus selected from the group consisting of influenza Type A viruses influenza Type B viruses and influenza type C viruses, wherein the fusion protein is capable of selectively binding anti-influenza antibodies. In one embodiment, the fusion protein comprises a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the sequence of a HA protein selected from the group consisting an H1 influenza virus HA protein, an H2 influenza virus HA protein, H3 influenza virus HA protein, an H4 influenza virus HA protein, an H5 influenza virus HA protein, an H6 influenza virus HA protein, an H7 virus influenza HA protein, an H8 influenza virus HA protein, an H9 influenza virus HA protein, an H10 influenza virus HA protein, an H11 influenza virus HA protein, an H12 influenza virus HA protein, an H13 influenza virus HA protein, an H14 influenza virus HA protein, an H15 influenza virus HA protein, and an H16 influenza virus HA protein, wherein the fusion protein is capable of selectively binding anti-influenza antibodies. In one embodiment, the fusion protein comprises a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the sequence of a HA protein from a virus listed in Table 2, wherein the fusion protein is capable of selectively binding anti-influenza antibodies. In one embodiment, the fusion protein comprises a ferritin protein of the present invention joined to amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38, wherein the fusion protein is capable of selectively binding anti-influenza antibodies. In one embodiment, the fusion protein comprises a ferritin protein of the present invention joined to amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38, wherein the fusion protein is capable of selectively binding anti-influenza antibodies.

In one embodiment the nanoparticle comprises a fusion protein comprising an amino acid sequence at least 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128, wherein the fusion protein is capable of selectively binding anti-influenza antibodies. In one embodiment the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128.

As used herein, the term contacting refers to the introduction of a sample being tested for the presence of anti-influenza antibodies to a nanoparticle of the present invention, for example, by combining or mixing the sample and the nanoparticle of the present invention, such that the nanoparticle is able to come into physical contact with antibodies in the sample, if present. When anti-influenza virus antibodies are present in the sample, an antibody/nanoparticle complex is then formed. Such complex formation refers to the ability of an anti-influenza virus antibodies to selectively bind to the HA portion of the fusion protein in the nanoparticle in order to form a stable complex that can be detected. Binding of anti-influenza virus antibodies in the sample to the nanoparticle is accomplished under conditions suitable to form a complex. Such conditions (e.g., appropriate concentrations, buffers, temperatures, reaction times) as well as methods to optimize such conditions are known to those skilled in the art. Binding can be measured using a variety of methods standard in the art including, but not limited to, agglutination assays, precipitation assays, enzyme immunoassays (e.g., ELISA), immunoprecipitation assays, immunoblot assays and other immunoassays as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Labs Press, 1989), and Harlow et al., Antibodies, a Laboratory Manual (Cold Spring Harbor Labs Press, 1988), both of which are incorporated by reference herein in their entirety. These references also provide examples of complex formation conditions.

As used herein, the phrases selectively binds hemagglutinin, selective binding to hemagglutinin, and the like, refer to the ability of an antibody to preferentially bind a HA protein as opposed to binding proteins unrelated to HA, or non-protein components in the sample or assay. An antibody that selectively binds HA is one that binds albumin but does not significantly bind other molecules or components that may be present in the sample or assay. Significant binding, is considered, for example, binding of an anti-HA antibody to a non-HA molecule with an affinity or avidity great enough to interfere with the ability of the assay to detect and/or determine the level of, anti-influenza antibodies in the sample. Examples of other molecules and compounds that may be present in the sample, or the assay, include, but are not limited to, non-HA proteins, such as albumin, lipids and carbohydrates.

In one embodiment, an anti-influenza virus antibody/nanoparticle complex, also referred to herein as an antibody/nanoparticle complex, can be formed in solution. In one embodiment an antibody/nanoparticle complex can be formed in which the nanoparticle is immobilized on (e.g., coated onto) a substrate. Immobilization techniques are known to those skilled in the art. Suitable substrate materials include, but are not limited to, plastic, glass, gel, celluloid, fabric, paper, and particulate materials. Examples of substrate materials include, but are not limited to, latex, polystyrene, nylon, nitrocellulose, agarose, cotton, PVDF (poly-vinylidene-fluoride), and magnetic resin. Suitable shapes for substrate material include, but are not limited to, a well (e.g., microtiter dish well), a microtiter plate, a dipstick, a strip, a bead, a lateral flow apparatus, a membrane, a filter, a tube, a dish, a celluloid-type matrix, a magnetic particle, and other particulates. Particularly preferred substrates include, for example, an ELISA plate, a dipstick, an immunodot strip, a radioimmunoassay plate, an agarose bead, a plastic bead, a latex bead, a cotton thread, a plastic chip, an immunoblot membrane, an immunoblot paper and a flow-through membrane. In one embodiment, a substrate, such as a particulate, can include a detectable marker. For descriptions of examples of substrate materials, see, for example, Kemeny, D. M. (1991) A Practical Guide to ELISA, Pergamon Press, Elmsford, N.Y. pp 33-44, and Price, C. and Newman, D. eds. Principles and Practice of Immunoassay, 2nd edition (1997) Stockton Press, NY, N.Y., both of which are incorporated herein by reference in their entirety.

In accordance with the present invention, once formed, an anti-influenza virus antibody/nanoparticle complex is detected. Detection can be qualitative, quantitative, or semi-quantitative. As used herein, the phrases detecting complex formation, detecting the complex, and the like, refer to identifying the presence of anti-influenza virus antibody complexed with the nanoparticle. If complexes are formed, the amount of complexes formed can, but need not be, quantified. Complex formation, or selective binding, between a putative anti-influenza virus antibody and a nanoparticle can be measured (i.e., detected, determined) using a variety of methods standard in the art (see, for example, Sambrook et al. supra.), examples of which are disclosed herein. A complex can be detected in a variety of ways including, but not limited to use of one or more of the following assays: a hemagglutination inhibition assay, a radial diffusion assay, an enzyme-linked immunoassay, a competitive enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, a particulate-based assay (e.g., using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, a BioCoreJ assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG=s Immunodot System, Fribourg, Switzerland), and an immunoblot assay (e.g., a western blot), an phosphorescence assay, a flow-through assay, a chromatography assay, a PAGe-based assay, a surface plasmon resonance assay, a spectrophotometric assay, and an electronic sensory assay. Such assays are well known to those skilled in the art.

Assays can be used to give qualitative or quantitative results depending on how they are used. Some assays, such as agglutination, particulate separation, and precipitation assays, can be observed visually (e.g., either by eye or by a machines, such as a densitometer or spectrophotometer) without the need for a detectable marker.

In other assays, conjugation (i.e., attachment) of a detectable marker to the nanoparticle, or to a reagent that selectively binds to the nanoparticle, aids in detecting complex formation. A detectable marker can be conjugated to the nanoparticle, or nanoparticle-binding reagent, at a site that does not interfere with ability of the nanoparticle to bind to an anti-influenza virus antibody. Methods of conjugation are known to those of skill in the art. Examples of detectable markers include, but are not limited to, a radioactive label, a fluorescent label, a chemiluminescent label, a chromophoric label, an enzyme label, a phosphorescent label, an electronic label; a metal sol label, a colored bead, a physical label, or a ligand. A ligand refers to a molecule that binds selectively to another molecule. Preferred detectable markers include, but are not limited to, fluorescein, a radioisotope, a phosphatase (e.g., alkaline phosphatase), biotin, avidin, a peroxidase (e.g., horseradish peroxidase), beta-galactosidase, and biotin-related compounds or avidin-related compounds (e.g., streptavidin or ImmunoPure7 NeutrAvidin).

In one embodiment, an antibody/nanoparticle complex can be detected by contacting a sample with a specific compound, such as an antibody, that binds to an anti-influenza antibody, ferritin, or to the antibody/nanoparticle complex, conjugated to a detectable marker. A detectable marker can be conjugated to the specific compound in such a manner as not to block the ability of the compound to bind to the complex being detected. Preferred detectable markers include, but are not limited to, fluorescein, a radioisotope, a phosphatase (e.g., alkaline phosphatase), biotin, avidin, a peroxidase (e.g., horseradish peroxidase), beta-galactosidase, and biotin-related compounds or avidin-related compounds (e.g., streptavidin or ImmunoPure7 NeutrAvidin).

In another embodiment, a complex is detected by contacting the complex with an indicator molecule. Suitable indicator molecules include molecules that can bind to the anti-influenza virus antibody/nanoparticle complex, the anti-influenza virus antibody, or the nanoparticle. As such, an indicator molecule can comprise, for example, a reagent that binds the anti-influenza virus antibody, such as an antibody that recognizes immunoglobulins. Preferred indicator molecules that are antibodies include, for example, antibodies reactive with the antibodies from species of individual in which the anti-influenza virus antibodies are produced. An indicator molecule itself can be attached to a detectable marker of the present invention. For example, an antibody can be conjugated to biotin, horseradish peroxidase, alkaline phosphatase or fluorescein.

The present invention can further comprise one or more layers and/or types of secondary molecules or other binding molecules capable of detecting the presence of an indicator molecule. For example, an untagged (i.e., not conjugated to a detectable marker) secondary antibody that selectively binds to an indicator molecule can be bound to a tagged (i.e., conjugated to a detectable marker) tertiary antibody that selectively binds to the secondary antibody. Suitable secondary antibodies, tertiary antibodies and other secondary or tertiary molecules can be readily selected by those skilled in the art. Preferred tertiary molecules can also be selected by those skilled in the art based upon the characteristics of the secondary molecule. The same strategy can be applied for subsequent layers.

Preferably, the indicator molecule is conjugated to a detectable marker. A developing agent is added, if required, and the substrate is submitted to a detection device for analysis. In some protocols, washing steps are added after one or both complex formation steps in order to remove excess reagents. If such steps are used, they involve conditions known to those skilled in the art such that excess reagents are removed but the complex is retained.

While the assays described thus far directly detect the antibody/nanoparticle complex, it is also possible to detect the complex using indirect methods. One example of an indirect assay is the hemagglutination inhibition (HAI) assay, which is a standard assay used to identify the levels of influenza virus in the serum of people thought to be infected with the virus. General methods for performing hemagglutinin inhibition assays are taught herein and are also disclosed in, for example, (see, for example, the WHO Reference on Animal Influenza Diagnosis and Surveillance, 2002, Department of Communicable Disease Surveillance and Response, World Health Organization).

The HAI assay is based on the fact that hemagglutinin protein on influenza virus is able to bind sialic acid molecules on red blood cells. Thus, when influenza virus is mixed with red blood cells, the virus and the red blood cells bind together forming a lattice (see FIG. 45), with the result that the lattice settles to the bottom of the reaction vessel (e.g., the well of an 96-well plate) forming a diffuse, red color over the entire surface. In contrast, if the reaction contains anything that interferes with binding of the HA protein to the sialic acid molecules on the red blood cells (such as anti-HA antibodies), formation of the lattice is prevented causing the red blood cells to fall out of solution and pool in the bottom of the container. In the latter case, the red blood cells form a characteristic red dot or “button” in the bottom of the well (see, for example, FIG. 46). In the HAI assay, a sample to be tested for the presence of anti-influenza antibodies is added to the influenza virus/red blood cell mixture (the assay mix) and the effect on button formation is observed. If the test sample contains anti-influenza antibodies, they will bind to the virus in the assay mixture, preventing, or disrupting, formation of the lattice causing the red blood cells to fall out of solution, pool in the well and form a button (or dot). The titer of the antibody present in the test sample is determined by counting how far the sample can be diluted before its hemagglutination inhibiting activity is lost. For example, if a sample diluted 1:1000 stil produces a dot nd the next dilution (1:2000) results in lattice formation (no dot) then the titer of antibody in the sample is 1:1000. Such methods of determining antibody titers are known to those skilled in the art.

A problem with the HAI assay is that it requires the use of live influenza virus, which due to the inherent dangers of growing such virus, requires the use of BSL2 and BSL3 facilities. However, such problems can be alleviated by using nanoparticles of the present invention in the HAI assay since they are fully recombinant and are made without the need to produce potentially dangerous live virus in eggs or in cell cultures. Moreover, because the fusion proteins making up the nanoparticles are recombinant, they offer the opportunity to test the effect of mutations in the HA protein that would otherwise inactivate viral replication. Thus, a HAI assay using nanoparticles of the present invention offers improvements and benefits not found in the currently available HAI assay.

Accordingly, in one embodiment, detection of an anti-influenza virus antibody/nanoparticle complex is conducted using a hemagglutination inhibition assay, wherein the hemagglutination inhibition assay is performed using nanoparticles of the present invention instead of influenza virus. More specifically, one embodiment of the present invention is a method for detecting anti-influenza virus antibodies in a sample, the method comprising:

-   -   a. contacting at least a portion of the sample with a         nanoparticle of the present invention and with red blood cells         comprising sialic acid molecules, to form a test mixture;     -   b. analyzing the test mixture for the presence of pooled red         blood cells (i.e., a button), wherein the presence of pooled red         blood cells (i.e., a button) in indicative of the presence         anti-influenza antibodies in the sample.

As has been described, the presence of anti-influenza antibodies in the test sample (at least at sufficient levels) causes the red blood cells to fall out of solution and pool, thereby forming a “button” (or dot) at the bottom of the vessel (e.g., ELISA well) housing the test mixture. Thus, formation of a button in the vessel containing the test mixture is indicative of the presence of anti-influenza antibodies in the test sample.

The level of red blood cell pooling (i.e., button formation) can be determined using any of the techniques known in the art for conducting hemagglutinin inhibition assays. In some embodiments, the level of button formation may be determined by simple visual inspection using the naked eye. In some embodiments, the level of button formation may be determined using a magnifying device such as, for example, a dissecting scope) or a microscope. In some embodiments, the level of button formation may be determined using a device such as, for example, a spectrophotometer or a refractometer. Methods of detecting or measuring button formation are known to those skilled in the art.

Any nanoparticle of the present invention can be used to practice hemagglutination inhibition assays of the present invention as long as it is capable of binding to sialic acid residues and to anti-influenza virus antibodies. Useful nanoparticles and methods of their production have been disclosed in detail herein. In a preferred embodiment, the nanoparticle comprises a fusion protein, wherein the fusion protein comprises at least 25, at least 50, at least 75, at least 100, or at least 150 contiguous amino acids from a monomeric ferritin subunit protein joined to at least one epitope from an influenza hemagglutinin protein (i.e., an HA-ferritin fusion protein) such that the nanoparticle comprises trimers of the influenza virus HA protein epitope on its surface, and wherein the fusion protein is capable of self-assembling into nanoparticles. In one embodiment the at least 25, at least 50, at least 75, at least 100, or at least 150 contiguous amino acids are from the region of a monomeric ferritin protein corresponding to the amino acid sequences of the Helicobacter pylori ferritin monomeric subunit that direct self-assembly of the monomeric subunits into the globular form of the ferritin protein. In one embodiment the at least 25, at least 50, at least 75, at least 100, or at least 150 contiguous amino acids are from SEQ ID NO:2, and are capable of directing self-assembly of the monomeric subunits into the globular ferritin protein. In one embodiment the at least 25, at least 50, at least 75, at least 100, or at least 150 contiguous amino acids are from amino acid residues 5-167 of SEQ ID NO:2, or from SEQ ID NO:5, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles.

In one embodiment the nanoparticle comprises a fusion protein comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identical to the amino acid sequence of a monomeric ferritin subunit that is responsible for directing self-assembly of the monomeric ferritin subunits into the globular form of the protein, wherein the fusion protein is capable of self-assembling into nanoparticles. In one embodiment, the fusion protein comprises a polypeptide sequence identical in sequence to a monomeric ferritin subunit. In one embodiment the fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identical to the amino acid sequence of a monomeric ferritin subunit from Helicobacter pylori, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles. In one embodiment the fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identical to the amino acid sequence of to a sequence selected from the group consisting of (1) amino acid residues 5-167 from SEQ ID NO:2 and (2) SEQ ID NO:5, wherein the HA-ferritin fusion protein is capable of self-assembling into nanoparticles. In one embodiment the fusion protein comprises a sequence selected from the group consisting of (1) amino acid residues 5-167 from SEQ ID NO:2 and (2) SEQ ID NO:5.

In one embodiment, the nanoparticle comprises a fusion protein comprising a ferritin protein of the present invention joined to at least one immunogenic portion of an HA protein from a virus selected from the group consisting of influenza type A viruses, influenza type B viruses and influenza type C viruses. In one embodiment the fusion protein comprises a ferritin protein of the present invention joined to at least one immunogenic portion of an HA protein selected from the group consisting of an H1 influenza virus HA protein, an H2 influenza virus HA protein, H3 influenza virus HA protein, an H4 influenza virus HA protein, an H5 influenza virus HA protein, an H6 influenza virus HA protein, an H7 virus influenza HA protein, an H8 influenza virus HA protein, an H9 influenza virus HA protein, an H10 influenza virus HA protein, an H11 influenza virus HA protein, an H12 influenza virus HA protein, an H13 influenza virus HA protein, an H14 influenza virus HA protein, an H15 influenza virus HA protein, and an H16 influenza virus HA protein. In one embodiment, the fusion protein comprises a ferritin protein of the present invention joined to at least one immunogenic portion of an HA protein from an influenza virus of the Victoria of Yamagata lineage. In one embodiment, the fusion protein comprises a ferritin protein of the present invention joined to at least one immunogenic portion of an HA protein from virus listed in Table 2. In, one embodiment the immunogenic portion comprises at least one epitope.

In one embodiment, the nanoparticle comprises a fusion protein comprising a ferritin protein of the present invention joined to an amino acid sequence that is a variant of an HA protein from a virus selected from the group consisting of influenza Type A viruses influenza Type B viruses and influenza type C viruses, wherein the fusion protein is capable of selectively binding anti-influenza antibodies. In one embodiment, the fusion protein comprises a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the sequence of a HA protein from a virus selected from the group consisting of influenza Type A viruses influenza Type B viruses and influenza type C viruses, wherein the fusion protein is capable of selectively binding anti-influenza antibodies. In one embodiment, the fusion protein comprises a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the sequence of a HA protein selected from the group consisting an H1 influenza virus HA protein, an H2 influenza virus HA protein, H3 influenza virus HA protein, an H4 influenza virus HA protein, an H5 influenza virus HA protein, an H6 influenza virus HA protein, an H7 virus influenza HA protein, an H8 influenza virus HA protein, an H9 influenza virus HA protein, an H10 influenza virus HA protein, an H11 influenza virus HA protein, an H12 influenza virus HA protein, an H13 influenza virus HA protein, an H14 influenza virus HA protein, an H15 influenza virus HA protein, and an H16 influenza virus HA protein, wherein the fusion protein is capable of selectively binding anti-influenza antibodies. In one embodiment, the fusion protein comprises a ferritin protein of the present invention joined to an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the sequence of a HA protein from a virus listed in Table 2, wherein the fusion protein is capable of selectively binding anti-influenza antibodies. In one embodiment, the fusion protein comprises a ferritin protein of the present invention joined to amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38, wherein the fusion protein is capable of selectively binding anti-influenza antibodies. In one embodiment, the fusion protein comprises a ferritin protein of the present invention joined to amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38, wherein the fusion protein is capable of selectively binding anti-influenza antibodies.

In one embodiment the nanoparticle comprises a fusion protein comprising an amino acid sequence at least 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128, wherein the fusion protein is capable of selectively binding anti-influenza antibodies. In one embodiment the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128.

Any red blood cell can be used to perform a HAI assay of the present invention as long as the red blood cells comprise sialic acid residues that are able to bind to influenza hemagglutinin protein. Examples of suitable red bloods cells include chicken red bloods cells, and turkey red bloods cells? Those skilled in the art are capable of identifying red blood cells useful for practicing the disclosed invention. Likewise, conditions suitable for formation of a complex between a nanoparticle and an anti-influenza antibody, if present, are known to those skilled in the art.

Because assays of the present invention can detect anti-influenza virus antibodies in a sample, including a blood sample, such assays can be used to identify individuals having anti-influenza antibodies. Thus, one embodiment of the present invention is a method to identify an individual having anti-influenza virus antibodies, the method comprising:

-   -   a. contacting a sample from an individual being tested for         anti-influenza antibodies with a nanoparticle of the present         invention; and,     -   b. analyzing the contacted sample for the presence of a         nanoparticle/antibody complex     -   wherein the presence of a nanoparticle/antibody complex         indicates the individual has anti-influenza antibodies.

Any of the disclosed assay formats can be used to conduct the disclosed method. Examples of useful assay formats include, but are not limited to, a hemagglutination inhibition assay, a radial diffusion assay, an enzyme-linked immunoassay, a competitive enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, a particulate-based assay (e.g., using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, a BioCoreJ assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG=s Immunodot System, Fribourg, Switzerland), and an immunoblot assay (e.g., a western blot), an phosphorescence assay, a flow-through assay, a chromatography assay, a PAGe-based assay, a surface plasmon resonance assay, a spectrophotometric assay, and an electronic sensory assay.

If no anti-influenza antibodies are detected in the sample, such a result indicates the individual does not have anti-influenza virus antibodies. The individual being tested may or may not be suspected of having antibodies to influenza virus. The disclosed methods may also be used to determine if an individual has been exposed to one or more specific type, group, sub-group or strain of influenza virus. To make such a determination, a sample is obtained from an individual that has tested negative for antibodies (i.e., lacked antibodies) to one or more specific type, group, sub-group or strain of influenza virus sometime in their past (e.g., greater than about 1 year, greater than about 2 years, greater than about 3 years, greater than about 4 years, greater than about 5 years, etc.). The sample is then tested for the presence of anti-influenza virus antibodies to one or more type, group, sub-group or strain, of influenza virus using a nanoparticle-based assay of the present invention. If the assay indicates the presence of such antibodies, the individual is then identified as having been exposed to one or more type, group sub-group or strain, of influenza virus sometime after the test identifying them as negative for anti-influenza antibodies. Thus, one embodiment of the present invention is method to identify an individual that has been exposed to influenza virus, the method comprising:

-   -   a. contacting at least a portion of a sample from an individual         being tested for anti-influenza antibodies with a nanoparticle         of the present invention; and,     -   b. analyzing the contacted sample for the presence or level of a         antibody/nanoparticle complex, wherein the presence or level of         antibody/nanoparticle complex indicates the presence or level of         recent anti-influenza antibodies;     -   c. comparing the recent anti-influenza antibody level with a         past anti-influenza antibody level;     -   wherein an increase in the recent anti-influenza antibody level         over the past anti-influenza antibody level indicates the         individual has been exposed to influenza virus subsequent to         determination of the past anti-influenza antibody level.

Methods of the present invention are also useful for determining the response of an individual to a vaccine. Thus, one embodiment is a method for measuring the response of an individual to an influenza vaccine, the method comprising:

-   -   a. administering to the individual a vaccine for influenza         virus;     -   b. contacting at least a portion of a sample from the individual         with a nanoparticle of the present invention;     -   c. analyzing the contacted sample for the presence or level of a         antibody/nanoparticle complex, wherein the presence or level of         antibody/nanoparticle complex indicates the presence or level of         recent anti-influenza antibodies     -   wherein an increase in the level of antibody in the sample over         the pre-vaccination level of antibody in the individual         indicates the vaccine induced an immune response in the         individual.

The influenza vaccine administered to the individual may, but need not, comprise a vaccine of the present invention, as long as the nanoparticle comprises an HA protein that can bind an anti-influenza antibody induced by the administered vaccine. Methods of administering influenza vaccines are known to those of skill in the art.

Analysis of the sample obtained from the individual may be performed using any of the disclosed assay formats. In one embodiment, analysis of the sample is performed using an assay format selected from the group consisting of, a hemagglutination inhibition assay, a radial diffusion assay, an enzyme-linked immunoassay, a competitive enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, a particulate-based assay (e.g., using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, a BioCoreJ assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG=s Immunodot System, Fribourg, Switzerland), and an immunoblot assay (e.g., a western blot), an phosphorescence assay, a flow-through assay, a chromatography assay, a PAGe-based assay, a surface plasmon resonance assay, a spectrophotometric assay, and an electronic sensory assay.

In one embodiment, the method includes a step of determining the level of anti-influenza antibody present in the individual prior to administering the vaccine. However, it is also possible to determine the level of anti-influenza antibody present in the individual from prior medical records, if such information is available.

While not necessary to perform the disclosed method, it may be preferable to wait some period of time between the step of administering the vaccine and the step of determining the level of anti-influenza antibody in the individual. In one embodiment, determination of the level of anti-influenza antibodies present in the individual is performed at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least one week, at least two weeks, at least three weeks, at least four weeks, at least two months, at least three months or at least six months, following administration of the vaccine.

The present invention also includes kits suitable for detecting anti-influenza antibodies. Suitable means of detection include the techniques disclosed herein, utilizing nanoparticles of the present invention. Kits may also comprise a detectable marker, such as an antibody that selectively binds to the nanoparticle, or other indicator molecules. The kit can also contain associated components, such as, but not limited to, buffers, labels, containers, inserts, tubings, vials, syringes and the like.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, and temperature is in degrees Celsius. Standard abbreviations are used.

Example 1 Design and Production of Ferritin-Based Nanoparticles Expressing Influenza Virus HA

This Example demonstrates the ability of HA-ferritin fusion proteins to form nanoparticles. Analysis of ferritin structure suggested that it was possible to insert a heterologous protein, specifically influenza virus HA, so that it mimics a physiologically relevant trimeric viral spike (FIG. 1A). Ferritin forms a nearly spherical particle consisting of 24 subunits arranged with octahedral symmetry around a hollow interior. The symmetry of the ferritin nanoparticles includes eight three-fold axes on the surface. The aspartic acid (Asp) at residue 5 near the NH₂ terminus is readily accessible, and the distance (28 Å) between each Asp5 on the three-fold axis is almost identical to the distance between the central axes of each HA2 subunit of trimeric HA (FIG. 1A, right).

Vector Construction.

The HA-ferritin fusion proteins were constructed by joining the ectodomain of A/New Calcdonia/20/1999 (1999 NC) HA to ferritin (FIG. 1B). Specifically, the gene encoding H. pylori nonheme iron-containing ferritin (GenBank NP_(—)223316) with a point mutation (N19Q) to abolish a potential N-linked glycosylation site was synthesized by PCR-based accurate synthesis (M. F. Bachmann, R. M. Zinkernagel, Neutralizing antiviral B cell responses. Annu Rev Immunol 15, 235-270 (1997)) using human-preferred codons. The human CD5 leader sequence and a serine-glycine-glycine (SGG) spacer were joined to the gene fragment encoding ferritin (residues 5-167) to generate a secreted protein. The plasmids encoding various influenza virus HAs, including A/South Carolina/1/1918 (1918 SC), GenBank AF117241; A/Puerto Rico/8/1934 (1934 PR8), UniProt P03452; A/Singapore/6/1986 (1986 Sing), GenBank AB038395; A/Beijing/262/1995 (1995 Beijing), GenBank AAP34323; A/New Calcdonia/20/1999 (1999 NC), GenBank AY289929; A/Solomon Islands/3/2006 (2006 SI), GenBank ABU99109; A/Brisbane/59/2007 (2007 Bris), GenBank ACA28844; A/California/04/2009 (2009 CA), GenBank ACP41105; A/Perth/16/2009 (H3 2009 Perth), GenBank ACS71642; B/Florida/04/2006 (B 2006 Florida), GenBank ACA33493 and their corresponding NAs with human preferred codons were synthesized as previously reported (C. J. Wei et al., Induction of broadly neutralizing H1N1 influenza antibodies by vaccination. Science 329, 1060-1064 (2010)). The gene fragments encoding HAs (residues HA1 1-HA2 174, H3 numbering) from 1999 NC HA, 2009 CA HA, 2009 Perth H3 and 2006 Florida B were amplified and joined to the ferritin gene fragment (residues 5-167) with an SGG linker to give rise to the HA-ferritin fusion gene. To produce soluble trimeric HA, the 1999 NC HA gene fragment (residues HA1 1-HA2 174, H3 numbering) was joined to a thrombin cleavage site followed by a foldon trimerization motif and a poly-histidine tag as described previously (A. S. Xiong et al., PCR-based accurate synthesis of long DNA sequences. Nat Protoc 1, 791-797 (2006)). Both full length and soluble forms of 1999 NC ΔStem (C. J. Wei et al., Induction of broadly neutralizing H1N1 influenza antibodies by vaccination. Science 329, 1060-1064 (2010)) and ΔRBS HA mutants were generated by introducing an N-linked glycosylation site at residues HA2 45 (145N/G47T) and HA1 190 (Q192T), respectively. The soluble form of 2007 Bris ΔRBS HA mutant was generated by introducing an N-linked glycosylation site at the same site. All genes were then cloned into mammalian expression vectors for efficient expression (C. J. Wei et al., Comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian H5N1 influenza virus. J Virol 82, 6200-6208 (2008)). Plasmids encoding the mAbs, CR6261 (D. C. Ekiert et al., Antibody recognition of a highly conserved influenza virus epitope. Science 324, 246-251 (2009)), CH65 (J. R. Whittle et al., Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin. Proc Natl Acad Sci USA 108, 14216-14221 (2011)) and a single-chain variable fragment F10 (J. Sui et al., Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol 16, 265-273 (2009)) were also synthesized as described by C. J. Wei et al., (Science 329, 1060-1064 (2010).

Protein Biosyntheses and Purifications.

To produce ferritin nanoparticles, HA-np and trimeric HA, the expression vectors were transfected into 293F cells (Invitrogen), a human embryonic kidney cell line using 293fectin (Invitrogen) according to the manufacturer's instructions. Matched NAs were co-transfected at 20:1 HA:NA (wt:wt). The cells were grown in Freestyle 293 expression medium (Invitrogen) and the culture supernatants were collected 4 days post-transfection by centrifugation and filtered through a 0.22 μm pore filter unit (Nalgene) to remove cell debris. The supernatants were concentrated with a 30 kDa molecular weight cut-off filter unit (Pall Corp.) and then buffer exchanged to a Tris buffer (20 mM Tris, 50 mM NaCl, pH 7.5 for ferritin nanoparticles; 20 mM Tris, 500 mM NaCl, pH 7.5 for HA-np). The ferritin nanoparticles were purified by ion-exchange chromatography using a HiLoad 16/10 Q Sepharose HP column (GE Healthcare). The HA-np were purified by affinity column chromatography using Erythrina cristagalli agglutinin (ECA, coral tree lectin; EY Laboratories, Inc.) specific for galactose β(1,4) N-acetylglucosamine. The ferritin nanoparticles and HA-np were further purified by size exclusion chromatography with a Superose 6 PG XK 16/70 column (GE Healthcare) in PBS. The peak fraction was collected and used for further studies. The molecular weights of the ferritin nanoparticle and HA-np were calculated based on two equations generated by least squares linear regression on a semi-log plot using gel filtration low and high molecular weight standards (Bio-Rad), respectively. The yield of 1999 NC HA-np is ˜4 mg liter⁻¹ and appears stable at 4° C. or frozen at −80° C. The trimeric HA proteins were purified as described by A. S. Xiong et at (Nat Protoc 1, 791-797 (2006)) with slight modifications. Briefly, HA proteins were first purified by affinity chromatography using Ni Sepharose HP resin (GE Healthcare), and then were separated by size exclusion chromatography with a HiLoad 16/60 Superdex 200 PG column (GE Healthcare). To remove the foldon trimerization motif and poly-histidine tag, HA proteins were digested with thrombin (EMD Chemicals, Inc.) (3 U mg ml⁻¹) overnight at 4° C. Undigested proteins were removed by passing over Ni Sepharose HP resin and the digested HAs were purified on a HiLoad 16/60 Superdex 200 PG column. All purified proteins were verified by SDS-PAGE. Protein purity and size distribution were examined by dynamic light scattering using a DynaPro system (Wyatt Technology). All human mAbs and a single-chain variable fragment were also produced in 293F cells and purified as described previously (C. J. Wei et al., Induction of broadly neutralizing H1N1 influenza antibodies by vaccination. Science 329, 1060-1064 (2010); W. P. Kong et al., Protective immunity to lethal challenge of the 1918 pandemic influenza virus by vaccination. Proc Natl Acad Sci USA 103, 15987-15991 (2006)). MAbs against 1999 NC HA were purified from hybridoma supernatants as previously described (C. J. Wei et al., Induction of broadly neutralizing H1N1 influenza antibodies by vaccination. Science 329, 1060-1064 (2010)).

Iodixanol-Based Gradient Centrifugation.

Alternatively, HA-np were purified by iodixanol gradient ultracentrifugation (FIG. 10) routinely used for virus and VLP purifications (C. J. Wei et al., Cross-neutralization of 1918 and 2009 influenza viruses: role of glycans in viral evolution and vaccine design. Sci Transl Med 2, 24ra21 (2010)). Fractions containing HA np were confirmed by SDS-PAGE and Western blotting using a mAb against 1999 NC HA.

Electron Microscopic Analysis.

Purified ferritin nanoparticles and HA-np were subjected to transmission electron microscopic analysis. The samples were negatively stained with phosphotungstic acid (ferritin nanoparticles) or ammonium molybdate (HA-np) and images were recorded on a Tecnai T12 microscope (FEI) at 80 kV with a CCD camera (AMT Corp.).

Analysis of HA-Ferritin np.

Among the various ferritins, Helicobacter (H.) pylori nonheme ferritin (K. J. Cho et al., The crystal structure of ferritin from Helicobacter pylori reveals unusual conformational changes for iron uptake. J Mol Biol 390, 83-98 (2009)) was selected as a prototype because of its highly divergent sequence compared to mammalian ferritins (FIG. 2), thus minimizing the likelihood of inducing autoimmunity after vaccination. The final purification step for recombinant HA-ferritin was size exclusion chromatography (FIG. 1C, left) and dynamic light scattering was used to confirm that both ferritin and HA-ferritin self-assembled into supramolecules with diameters of 14.61 and 37.23 nm, respectively (FIG. 1C, middle). HA-ferritin and ferritin subunits from these nanoparticles migrated at the expected respective molecular weights of 85 and 17 kDa by SDS-PAGE compared to 68 kDa for purified HA (FIG. 1C, right). While the morphology of the ferritin nanoparticles was smooth, as visualized by transmission electron microscopy (TEM), HA-ferritin formed np that exhibited clearly visible spikes around the spherical core (FIG. 1D, Ferritin np vs. HA-np). Remarkably, the placement of these spikes clearly illustrated the octahedral symmetry of the HA-np design. Octahedral two-, three- and four-fold axes were distinctly observed in the TEM image (FIG. 1E, right). These data demonstrated the formation of HA spikes on self-assembling HA-ferritin nanoparticles. More importantly, this design enabled HAs from different subtypes or influenza B viruses to be readily joined to a ferritin core without substantial modification.

Example 2 Antigenicity and Immunogenicity of HA-np in Mice

To verify the antigenicity of the HA spikes on the np, HA-ferritin np were analyzed for their ability to react with anti-HA head ab and a conformation-dependent monoclonal ab (mAb), CR6261, that recognizes a highly conserved structure in the trimeric HA stem and neutralizes diverse influenza A group 1 viruses D. C. Ekiert et al., Antibody recognition of a highly conserved influenza virus epitope. Science 324, 246-251 (2009)), using ELISA and a virus neutralization assay.

Analysis by ELISA.

Purified trimeric HA, HA-np, and TIV (2 μg of H1 HA ml⁻¹), ferritin nanoparticles (0.68 μg ml⁻¹ for FIG. 3 or 2 μg ml⁻¹ for the rest), mouse liver ferritin (2 μg ml⁻¹, Alpha Diagnostic International, Inc.), ΔStem and ΔRBS HA trimer (2 μg ml⁻¹) were coated (100 μl/well) onto MaxiSorp™ plates (Nunc) and the wells were probed with the anti-HA mAbs, anti-mouse liver ferritin IgG (Alpha Diagnostic International, Inc.) or immune sera followed by peroxidase-conjugated secondary antibodies (anti-mouse IgG and anti-human IgG, SouthernBiotech; anti-ferret IgG, Rockland Immunochemicals, Inc.). The wells were developed using a SureBlue chromogen (KPL) and the reaction was stopped by adding 0.5 M sulfuric acid. For the ELISA-based competition assay, HA trimer (2 μg ml⁻¹) was coated onto the plates. Plates were incubated with an anti-stem mAb, CR6261 (8 μg ml⁻¹) or an isotype control Ab, VRC01 (8 μg ml⁻¹) (Z. Y. Yang et al., Immunization by avian H5 influenza hemagglutinin mutants with altered receptor binding specificity. Science 317, 825-828 (2007); X. Wu et al., Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 329, 856-861 (2010)) before adding serially diluted pre-absorbed ferret immune sera. The wells were probed with anti-ferret IgG and developed as described above. Absorbance at 450 nm was measured by SpectraMax M2e (Molecular Devices). The endpoint titers were determined by calculating the intersection of the observed binding curve and the absorbance threshold (four times background).

Neutralization Assays.

HA/NA-pseudotyped lentiviral vectors encoding luciferase were used. Immune sera used for the assay were pretreated with RDE as described above. Pre-titrated pseudotyped viruses (Gag p24≈6.25 ng ml⁻¹) were incubated with serially diluted sera for 20 minutes at room temperature and added to 293A cells (10,000 cells/well in a 96-well plate; 50 μl/well; in triplicate). Plates were then washed and replaced with fresh media 2 hours later, and luciferase activity was measured after 24 hours. For the protein competition assay, neutralizing activity of the mAbs F10, CR6261 or immune sera was measured in the presence of competitor proteins, trimeric HA (WT, ΔStem or ΔRBS), HA-np, ferritin nanoparticles or irrelevant protein (HIV-1 gp120) at final concentration of 20 and 25 μg ml⁻¹ for mAbs and immune sera, respectively. The HA-np was able to bind to anti-head or anti-stem mAbs with affinities similar to trimeric HA or trivalent inactivated vaccine (TIV) containing the same 1999 NC HA at equimolar concentrations of HA, in contrast to a ferritin nanoparticle control (FIG. 3A). Analogous to trimeric HA, the HA-np also blocked neutralization by CR6261 and another stem-directed mAb, F10 (4 J. Sui et al., Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol 16, 265-273 (2009)) (FIG. 3B). These results indicated that HA molecules on the HA-np antigenically resembled the physiological trimeric viral spike.

Example 3 Immunogenicity of HA-Ferritin np In Vivo

This Example demonstrates the ability of HA-ferritin np of the present invention to elicit neutralizing antibodies.

To assess the immunogenicity of the HA-ferritin np in vivo, mice were immunized twice with HA-np or TIV's from the 2006-2007 season, with HAs from A/New Calcdonia/20/1999 (H1N1), A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2504/04 (type B), or from the 2011-2012 season, with HAs from A/California/07/09-like (H1N1), A/Perth/16/09 (H3N2) and B/Brisbane/60/08 (type B). Briefly, female BALB/c mice (6-8 weeks old; Charles River Laboratories) were immunized (5 mice/group) intramuscularly with 5 or 0.5 μg (1.67 or 0.17 μg of H1 HA) of TIV, 2.24 or 0.22 μg (1.67 or 0.17 μg of HA) of HA-np or 0.57 μg of ferritin nanoparticles (equimolar to 2.24 μg of HA-np) in 100 μl of PBS or in 100 μl of 50% (v/v) mixture of Ribi adjuvant (Sigma) in PBS at weeks 0 and 3. A group of BALB/c mice (n=4) was immunized with 20 μg of trimeric HA (thrombin cleaved) in 100 μl of 50% (v/v) mixture of Ribi adjuvant in PBS at weeks 0 and 4. For the experiment using trivalent HA-np, mice were immunized (n=5) with 6.72 μg (1.67 μg of each HA component) of trivalent HA-np in 100 μl of 50% (v/v) mixture of Ribi adjuvant in PBS at weeks 0 and 3. Blood samples were collected prior to the first dose, and at 2 weeks after each immunization.

The resulting antibody titers were determined as described in Example 2. The HA-np induced significantly higher HAI titers than TIV (FIG. 4A, left; p<0.0001), and a similar effect was observed in the neutralization assay and ELISA (FIG. 4A, middle and right; p<0.0001). For example, neutralization titers elicited by HA-np as assessed by the concentration of ab needed to inhibit viral entry by 90% (IC₉₀) were ˜34 times higher than TIV (FIG. 4A, middle). Because higher titers were observed in groups with the adjuvant Ribi, further comparisons were performed with this adjuvant. Neutralization against a panel of H1N1 strains revealed not only increased potency but also enhanced breadth stimulated by HA-np compared with TIV or trimeric HA (FIG. 4B). Neutralization against two highly divergent H1N1 viruses, A/Puerto Rico/8/1934 (1934 PR8) and A/Singapore/6/1986 (1986 Sing) were only observed in mice immunized with the HA-np, and the titer against the contemporary virus A/Brisbane/59/2007 (2007 Bris) was more than one log higher in mice immunized with HA-np than with TIV (FIG. 4B).

To assess whether the preexisting immune responses to ferritin nanoparticles or to other HA subtypes would attenuate the immunogenicity of the subsequent immunization of HA-np, mice were pre-immunized with either H3 (A/Perth/16/09, 2009 Perth) HA-np or empty ferritin nanoparticles to elicit anti-H3 HA and/or anti-H. pylori ferritin immune responses (FIG. 5A). These animals were then immunized with H1 (1999 NC) HA-np. Comparable HAI, IC₉₀ neutralization and ELISA titers against 1999 NC HA were observed in naïve animals as well as in groups pre-immunized with H3 HA-np or empty ferritin nanoparticles (FIG. 5B). These results indicated that preexisting anti-H. pylori ferritin immunity did not diminish the HA-specific ab response.

Example 4 Lack of Autoreactivity of H. pylori Ferritin Nanoparticles

This Example demonstrates analyzes the ability of HA-ferritin np of the present invention to elicit an auto-immune response against autologous ferritin in mice.

Although the overall structural architecture and physiological functions of ferritin are conserved across organisms, murine ferritin has only 27% amino acid sequence identity to H. pylori ferritin. This homology nonetheless raised the possibility that immunization with H. pylori ferritin in mice might abrogate immune tolerance and induce autoimmunity. To address this concern, CD4, CD8 T-cell and ab responses against both murine and H. pylori ferritins were analyzed by intracellular cytokine staining (ICS) and ELISA in mice immunized with HA-np. ELISAs were performed according to the procedure in Example 2. For intracellular cytokine analysis, CD4⁺ and CD8⁺ T-cell responses were evaluated for interferon-γ (IFN-γ), tumor necrosis factor α (TNFα), and interleukin-2 (IL-2) as described by T. Zhou et al. (Science 329, 811-817 (2010)). Individual peptide pools (15-mer overlapping by 11 residues, 2.5 μg ml⁻¹ for each peptide) covering H. pylori ferritin or mouse ferritin light and heavy chains were used to stimulate cells. After stimulation, cells were fixed, permeabilized and stained using anti-mouse CD3, CD4, CD8, IFN-γ, TNFα and IL-2 mAbs (BD Pharmingen) together with aqua blue dye for live/dead stain (Invitrogen). The data were collected by LSR II Flow Cytometer (BD Biosciences) and IFN-γ-, TNFα- and IL-2-positive cells in the CD4⁺ and CD8⁺ cell populations were analyzed with FlowJo software (Tree Star).

Although an increase in the ICS staining of CD4⁺ T cells stimulated with H. pylori ferritin peptides (FIG. 4C, top left) was observed, no increases in the CD4⁺ and CD8⁺ ICS responses were seen with murine ferritin peptide stimulation (FIG. 4C, bottom left and middle). In addition, while high titers (>10⁶) of anti-H. pylori ferritin abs were detected in ferritin nanoparticle- and HA-np-immune sera, abs to mouse ferritin were undetectable (FIG. 4C, right). These results demonstrate that HA-ferritin np of the present invention do not elicit autoreactivity to autologous ferritin in mice.

Example 5 Generation of Trivalent HA-np and Immunogenicity in Mice

The Example analyzes whether multivalent HA-np were similar in immunogenicity to monovalent np.

HA-np expressing HAs from H1 (A/California/04/09, 2009 CA), H3 (2009 Perth) or influenza B (B/Florida/04/06, 2006 FL) were generated. The 2009 CA (H1)-, 2009 Perth (H3)- and 2006 FL (type B)-HA-np self-assembled and displayed the same morphology observed for 1999 NC HA-np (FIG. 6A). Trivalent HA-np were generated by combining three monovalent HA-np, and their immunogenicity was compared to a seasonal TIV containing the same H1 and H3 strains and a mismatched type B (B/Brisbane/60/08). HAI titers against homologous H1N1 and H3N2 viruses were significantly increased in animals immunized with trivalent HA-np relative to TIV-immunized animals (FIG. 6B; p=0.0125 and 0.0036, respectively). When compared to animals immunized with the corresponding monovalent HA-np, HAI titers against 2009 CA (H1) and 2009 Perth (H3) induced by trivalent HA-np were comparable (FIG. 6B). These results demonstrate that no substantial antigenic competition between H1 and H3 HA-np was observed with a trivalent HA-np vaccine.

Example 6 Cross-Protective Immunity Elicited by HA-np in Ferrets

This Example demonstrates that vaccination of ferrets with 1999 NC HA-np elicits a protective immunity similar to that observed in human disease.

Male Fitch ferrets (6 months old; Triple F Farms), seronegative for exposure to H1N1, H3N2 and type B influenza viruses, were housed and cared for at BIOQUAL, Inc. (Rockville, Md.). Prior to study start, a temperature transponder (Biomedic Data Systems, Inc.) was implanted into the neck of each ferret. Ferrets were immunized (6 ferrets/group) intramuscularly with 500 μl of PBS, 7.5 μg (2.5 μg of H1 HA) of TIV or 3.35 μg (2.5 μg of HA) of HA-np in 500 μl of 50% (v/v) mixture of Ribi adjuvant in PBS at weeks 0 and 4. Blood was collected 3 and 2 weeks after the first and the second immunization, respectively.

Three weeks after the first immunization, all ferrets receiving HA-np generated protective HAI titers against homologous H1 1999 NC virus (>1:40), while only 50% (3/6) of TIV-immunized ferrets induced HAI titers greater than 1:40 (FIG. 7A, left; p=0.0056). The same trend was also observed for both neutralization and anti-HA ab titers (FIG. 7A, middle and right; p=0.0047 and p=0.0045, respectively), documenting the superior potency of HA-np in a second species. After boosting, the HAI and IC₉₀ neutralization titers of the HA-np-immune sera were ˜10-fold higher than those of TIV-immunized ferrets (FIG. 7A, left and middle; 457±185 vs. 5760±1541, p=0.0066, and 598±229 vs. 5515±1074, p=0.0012, respectively). A similar enhancement in HA-np vs. TIV immunization was also observed by ELISA titers (FIG. 7A, right; p=0.0038). Remarkably, a single immunization with HA-np induced immune responses comparable to two immunizations with TIV (FIG. 7A).

To determine whether HA-np could confer protection against an unmatched H1N1 virus, five weeks after the last immunization ferrets immunized with 1999 NC HA-np or TIV containing the same H1 HA were challenged with 10^(6.5) EID₅₀ of 2007 Bris virus. (1999 NC and 2007 Bris viruses are 8 years apart and their antigenic characteristics are sufficiently different to require the production of two different vaccines to confer protection in humans.) The virus was expanded in embryonated chicken eggs from a seed stock obtained from CDC (Atlanta, Ga.) and has a titer of 10^(6.5) EID₅₀ ml⁻¹. The virus stock was inoculated intranasally into ferrets, which had been anesthetized with ketamine/xylazine, in a volume of 500 μl per nostril. The ferrets were observed for clinical signs twice daily and weight and temperature measurements recorded daily by technicians blind to the treatment groups. Nasal washes were obtained on days 1, 3 and 5 and infectious viral titers were determined by TCID₅₀ assay using MDCK cells as described previously (C. J. Wei et al., Induction of broadly neutralizing H1N1 influenza antibodies by vaccination. Science 329, 1060-1064 (2010)).

Ferrets immunized with HA-np showed a significant reduction in viral shedding beginning 1 day after challenge compared to the sham control group (FIG. 7B, left; p=0.0259). At the same time point, no reduction in viral shedding was seen in the TIV-immunized group. Four of six animals immunized with HA-np had no detectable viral load after 3 days and by day 5, all animals in this group cleared the virus, while all animals in the sham control group still had detectable virus (FIG. 7B). In addition, HA-np-immunized ferrets suffered less body weight loss compared to the TIV-immunized and sham control groups (FIG. 7B, right). These results demonstrate faster virus clearance in ferrets immunized with HA-np than with TIV and further demonstrate that HA-np effectively induced cross-protective immunity in vaccinated ferrets.

Example 7 Induction of Two Types of Neutralizing abs (nAbs) in Ferrets

This Example demonstrates the breadth and specificity of nAbs in ferret immune sera.

IC₅₀ neutralization titers against 1986 Sing, A/Beijing/262/1995 (1995 Beijing), A/Solomon Islands/3/2006 (2006 SI) and 2007 Bris were significantly higher in animals immunized with HA-np compared to immunization with TIV (FIG. 8A, left). This enhanced breadth was due not only to a quantitative increase in overall ab titer (˜9-fold against matched virus) but also reflected a qualitative difference in the types of abs elicited (>40-fold enhancement against an unmatched strain). To determine whether the cross-reactivity induced by HA-np was due to nAbs to the conserved HA stem epitope, ferret immune sera were pre-absorbed with cells expressing a stem mutant (ΔStem) HA to remove non-stem directed antibodies. Briefly, ferret immune sera taken 2 weeks after the second immunization were subjected to the assay. The plasmids encoding for ΔStem and ΔRBS HAs were transfected into 293F cells. Three days after transfection, the cells were analyzed by flow cytometry to confirm expression of HA on the cell surface and used for serum absorption. One ml of the immune sera diluted at 1:100 and 1:1,000 was incubated with 100 μl of pre-washed ΔStem and ΔRBS HA-expressing 293F cell pellets, respectively. After incubating for 1 hour at 4° C., supernatants were harvested by centrifugation and binding to WT and mutant HAs was examined by ELISA previously described (C. J. Wei et al., Induction of broadly neutralizing H1N1 influenza antibodies by vaccination. Science 329, 1060-1064 (2010)). The ΔStem HA-pre-absorbed sera were also used for competition ELISA.

Stem-specific abs were detected in HA-np-immunized ferrets (6/6) in greater frequency and magnitude than TIV-immune ferrets (2/6) (FIG. 8B, left; p=0.0056). Moreover, binding of these pre-absorbed sera to HA was inhibited by CR6261 mAb (FIG. 8B, right; p=0.0019), further documenting the specificity of HA-np immune sera to the stem epitope. The HAI titers against heterologous 2007 Bris virus were also significantly higher in ferrets immunized with HA-np (6/6, 1:80-1:640) than with TIV (3/6, 1:40-1:80) (FIG. 8A, right; p=0.0054). Interestingly, in contrast to a previous study in which DNA prime/TIV boost was used to elicit anti-stem broadly neutralizing abs (bnAbs) (C. J. Wei et al., Induction of broadly neutralizing H1N1 influenza antibodies by vaccination. Science 329, 1060-1064 (2010)), sera from animals immunized with HA-np showed HAI ab titers against a highly divergent 1934 PR8 strain, with titers ≧1:40 in all ferrets. However, no HAI titers against 1934 PR8 were detected in TIV-immunized ferrets (FIG. 8A, right). These data suggested that the HA-np vaccine might elicit another class of nAb directed towards the conserved RBS in the HA head.

To determine whether HA-np elicited abs against RBS, an RBS mutant HA (ΔRBS) was generated by introducing a glycosylation site in the sialic acid binding pocket at residue 190 (FIG. 9) (D. Lingwood et al., Structural and genetic basis for development of broadly neutralizing influenza antibodies. Nature, in press). Ferret immune sera were absorbed with ΔRBS HA-expressing cells to remove abs to HA outside of this region and tested for binding against WT or ΔRBS HA. RBS-directed abs were detected with titers of >1:2,000 in all HA-np-immunized ferrets, but only 1 out of 6 ferrets that received TIV (FIG. 8B, middle).

To define the relative contributions of these stem and RBS abs to the breadth of neutralization, neutralization assays were performed in the presence of competitor proteins: WT, ΔStem or ΔRBS HA. In the presence of excess ΔStem HA, only stem-directed abs can neutralize viruses; similarly, ΔRBS HA interferes with all antibodies in the serum except those proximal to the RBS. The relative contribution of stem- and RBS-directed neutralization was measured as activity remaining in the presence of the respective competitor HA. For example, with 2007 Bris, ΔRBS HA only partially inhibited neutralization, while either WT or ΔStem HA almost completely abolished the neutralization activity of the sera; hence, the neutralization against 2007 Bris was due almost entirely to RBS-directed abs (FIG. 8C). Four H1N1 strains were tested in this assay. The pattern of neutralization inhibition varied by strain. Neutralization of 1999 NC or 2007 Bris was mediated predominantly by RBS-directed abs. However, neutralization of 1986 Sing was due mainly to stem-directed abs. Interestingly, the neutralization of 1995 Beijing was more complex. Both stem- and RBS-directed abs contributed to neutralization of this virus (FIG. 8C).

These results demonstrate that HA-np induce both known types of bnAbs-stem-directed and RBS-directed. Together, these abs contribute to the breadth and potency of the immune sera elicited by HA-np. The synergy between them explains mechanistically the observed superior efficacy of the HA-np vaccine and decreases the likelihood of viral escape mutations from either antibody alone.

Taken together the above-disclosed Examples demonstrate that a ferritin-based nanoparticle is able to present trimeric HA in its native fold, rigidly and symmetrically, with sufficient spacing to ensure optimal access to potential bnAbs directed to the stem. They also demonstrate that the nanoparticles have enhanced immunogenicity and an expanded neutralization breadth to both stem and RBD antibodies.

Example 8 Immunization of Mice and Ferrets Using a Tetravalent Vaccine

This Example demonstrates the ability of a multivalent vaccine to elicit an immune response against several strains and sub-types of influenza virus.

The ability of a pan-group 1 vaccine to stimulate neutralizing antibodies against a variety of influenza viruses was tested in mice and ferrets using a protocol similar to that described in Example 1, and outlined in FIG. 11. Briefly, a pan-group 1 HA-ferritin np vaccine was produced by combining four different monovalent HA-ferritin np vaccines. Specifically, HA-ferritin np, each expressing either H1 A/NC/20/1999, H1 A/CA/04/2009, H2 A/Singapore/1/1957 or H5 A/Indonesia/05/2005, were combined to produce a single vaccine containing all four HA proteins. Mice were immunized twice in a four week interval using 6.8 ug total of the pan-group 1 vaccine (1.7 ug of each HA-ferritin np) in Ribi. Ferrets were immunized twice in a four week interval using 10 ug total of the pan-group 1 vaccine (2.5 ug of each HA-ferritin np) in Ribi. Blood was obtained from the immunized animals and the titer of neutralizing antibodies against various influenza viruses measured. The results of this analysis are shown in FIGS. 12-14. Immunized ferrets were also challenged with either influenza A/Brisbane/59/2007 Brisbane (H1N1) (2207 Bris) (FIG. 15) or influenza A/Mexico/2009 (H1N1) (2009 Mex) (FIG. 16) and the resulting virus titers measured on day 3 and 5 post-challenge.

Example 9 Design and Construction HA-Ferritin Stem-Region Fusion Proteins

This Example demonstrates the construction of HA-ferritin proteins and nanoparticles that present the stem region of the influenza HA protein.

As illustrated in FIG. 17, the stem region of the influenza HA protein is highly conserved among different influenza strains, and possesses a site of vulnerability for Group 1 viruses. Thus, a vaccine that elicits neutralizing antibodies against the stem region of the influenza HA protein should be broadly neutralizing. A nanoparticle displaying the stem region of the influenza stem region was constructed as a vaccine.

Design of an HA-Stabilized Stem Fusion Protein

An HA-stabilized stem fusion protein (HA SS) was constructed as follows: residues 43-313 of the head domain of HA1 were replace with a Gly-Trp-Gly linker. The membrane distal end of HA2 (residues 59 to 93) was replaced by an HIV-1 Bal gp41 HR2 helix followed by a six residue glycine-rich linker (Asn-Gly-Thr-Gly-Gly-Gly-Ser-Gly) and the gp41 HR1 helix. The HR1 helix of gp41 was added in frame with helix C of HA2 so as to generate a long central chimeric helix. The resulting six helix bundle sitting atop the modified hemagglutinin stem provides stability to the SS trimer in lieu of the missing head residues. A schematic of the resulting protein is shown in FIG. 18A, while a ribbon diagram is shown in FIG. 18B. A second trimerization domain consisting of a 28 residue T4 foldon domain was joined to the membrane proximal C-terminus of HA2. The HA 55-ferritin nanoparticle (HA SS-np) protein was generated by joining residue 174 (H3 numbering) of HA SS to H. pylori ferritin (residues 5-167) with a Ser-Gly-Gly linker.

In constructing HA-SS fusion proteins, genes encoding wild-type HA proteins (A/Puerto Rico/8/1934 (H1 1934 PR8), A/Singapore/6/1986 (H1 1986 Sing), A/New Calcdonia/20/1999 (H1 1999 NC), A/Brisbane/59/2007 (H1 2007 Bris), A/Vietnam/1203/2004 (H5 2004 VN), A/Canada/720/05 (H2 2005 CAN), A/Hong Kong/1/1968 (H3 1968 HK), A/Hong Kong/1073/1999 (H9 1999 HK) and their corresponding NAs, H1 NC 99 SS, RSC3 HIV gp120 control protein, and all Abs (CR6261, F16v3, and VRC01) were synthesized with human preferred codons as previously described (Wei et al. Science 2010, 329(5995):1060-4). Helicobacter pylori nonheme iron-containing ferritin (GenBank NP_(—)223316) with a point mutation (N19Q) to abolish a potential N-linked glycosylation site was synthesized by PCR-based accurate synthesis (Xiong et al. Nat Protoc 2006, 1(2):791-797) using human-preferred codons. Coding sequences for the human CD5 leader sequence and a serine-glycine-glycine (SGG) spacer were joined to the gene fragment encoding ferritin (residues 5-167) to generate a secreted protein. HA and HA SS— np fusion proteins were generated by overlap PCR by joining the HA ectodomains at residue HA2 174 (H3 numbering) to H. pylori ferritin (residues 5-167) with a Ser-Gly-Gly linker. Stem mutant probes Δstem (glycosylation insertion into the CR6261 binding epitope at position 45 in HA2; H3 numbering) which prevent binding at the conserved H1 stem epitope were generated using site directed mutagenesis. Genes encoding these proteins were cloned into a CMVR plasmid backbone for efficient mammalian cell expression.

Protein Expression and Purification

Plasmids encoding soluble proteins were transfected (HA ectodomain genes were cotransfected with the corresponding NA encoding plasmids) into the human embryonic kidney cell line 293F and isolated from expression supernatants 72-96 hrs post-transfection. All HA and HA SS trimeric proteins were purified first by metal chelation affinity chromatography and then by size exclusion chromatography as previously described (Wei et al. J Virol. 2008, 82(13):6200-8). IgG Abs were purified using a Protein G affinity column (GE Healthcare). The HA- and HA SS-np were purified by affinity column chromatography using Erythrina cristagalli agglutinin (ECA, coral tree lectin; EY Laboratories, Inc.) specific for galactose β(1,4) N-acetylglucosamine and Galanthus nivalis agglutinin (GNA, snowdrop lectin; EY Laboratories, Inc.) specific for α(1,3) and α(1,6) linked high mannose structures, respectively. HA- and HA SS-np were further purified by size exclusion chromatography with a Superose 6 PG XK 16/70 column (GE Healthcare) in PBS (FIG. 19).

HA SS-Ferritin Characterization.

HA SS-ferritin np were visualized by electron microscopy. Briefly, purified HA SS-np were negatively stained with phosphotungstic acid and ammonium molybdate, respectively, and images were recorded on a Tecnai T12 microscope (FEI) at 80 kV with a CCD camera (AMT Corp.). The results of this analysis are shown in FIG. 20. IN addition, the ability of purified HA SS and HA SS-np to bind to monoclonal Abs CR6261 and FI6v3 (1.7×10⁻⁴ to 10 μg/mL) was characterized by ELISA. HA and HIV gp120 proteins served as controls. Ab binding was detected by peroxidase-conjugated goat anti-human IgG. The results of this analysis, which are shown in FIG. 21, demonstrate that HASS-ferritin is antigenically similar to HA protein.

Example 10 Immune Response to HA SS-Ferritin Nanoparticles

This Example demonstrates the immune response generated in animals following immunization with HA SS-ferritin np.

BALB/c mice were immunized twice intramuscularly with protein (2 or 10 μg each) formulated with Ribi adjuvant system (Sigma) at a 3 week interval. Mice received either homologous (HA SS-np prime and boost) or heterologous (HA-np prime and HA SS-np boost) immunizations. Ferrets were immunized three times intramuscularly with HA SS-np (10 μg each) formulated with Ribi adjuvant system (Sigma) at weeks 0, 4 and 14. Serum was collected from animals 2 weeks after each immunization and 1 week prior to the first immunization and heat inactivated (30 min at 56° C.).

Pre- and post-immune sera from immunized mice and ferrets were assayed for binding to HA and HA SS by ELISA. Briefly, sera were serially diluted (diluted 50 to 2.3×10⁶) and assayed for reactivity to soluble trimeric HA and HA SS proteins, as well as control proteins (200 ng/well with molar equivalents plated according to HA SS). Binding was detected by peroxidase conjugated anti-mouse or anti-ferret IgG, respectively. Endpoint dilutions were determined from nonlinear fit dose-response curves using a detection limit of 2× background absorbance. The result from this analysis are shown in FIG. 22 and demonstrate that stem specific cross-reactive antibodies which recognize the conserved stem-epitope are elicited by HA SS-np vaccination.

Sera were also analyzed for neutralization of pseudotyped recombinant lentiviruses expressing wild-type HA with the corresponding NA with a luciferase reporter gene as previously described (Wei et al. Science 2010, 329(5995):1060-4) following pretreatment with receptor-destroying enzyme (RDE II; Denka Seiken Co., Ltd.). Psuedotype neutralization competition of ferret serum was performed by incubating serially diluted serum in the presence of either H1 1999 NC SS, H1 1999 NC SS Δstem probe or gp120 control (10 μg/mL) for 1 hr (RT) before addition to pseudotyped recombinant lentiviruses and assaying for neutralization. The results from this analysis are shown in FIG. 23 and demonstrate that vaccination with HA SS-np elicits neutralizing antibodies against various group-1 strains.

Example 11 Immune Response to HA SS-Ferritin Heterologous Immunization Boost

This example demonstrates that HA SS-np can be utilized to boost antibodies directed to the conserved stem epitope.

BALB/c mice were immunized twice intramuscularly with heterologous ferritin proteins (HA-np prime and HA SS-np boost; 2 μg each) formulated with Ribi adjuvant system (Sigma) at a 3 week interval. Serum was collected from animals 2 weeks after each immunization and 1 week prior to the first immunization and heat inactivated (30 min at 56° C.).

Pre- and post-immune sera from immunized mice were assayed for binding to HA and HA SS by ELISA. Briefly, sera were serially diluted (diluted 50 to 2.3×10⁶) and assayed for reactivity to soluble trimeric HA and HA SS proteins, as well as control proteins (200 ng/well with molar equivalents plated according to HA SS). Binding was detected by peroxidase conjugated anti-mouse or anti-ferret IgG, respectively. Endpoint dilutions were determined from nonlinear fit dose-response curves using a detection limit of 2× background absorbance. The results from this analysis are shown in FIG. 22 and demonstrate that cross-reactive stem-epitope specific antibodies are being elicited.

Sera were also analyzed for neutralization of pseudotyped recombinant lentiviruses expressing wild-type HA with the corresponding NA with a luciferase reporter gene as previously described (Wei et al. Science 2010, 329(5995):1060-4) following pretreatment with receptor-destroying enzyme (RDE II; Denka Seiken Co., Ltd.). The results from this analysis are shown in FIG. 24 and demonstrate that mice which have preexisting stem antibodies titers can be boosted with HA SS-np.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims.

Example 12 Use of HA-Ferritin Nanoparticles in a Hemagglutination Inhibition Assay

This Example demonstrates the ability of HA-ferritin nanoparticles of the invention to agglutinate red blood cells as well as the utility of such nanoparticles in performing hemagglutination inhibition assays.

To compare the ability of HA-ferritin nanoparticles to agglutinate red blood cells, agglutination assays were performed using either inactivated influenza virus of HA-ferritin nanoparticles. Briefly, using 96-well plates, inactivated vaccine and HA-ferritin nanoparticles were each serially diluted in PBS and incubated with 0.5% chicken RBCs (vol/vol using PBS) for 30 minutes at room temperature. Hemagglutination mediated through sialic acid moiety on RBC surface and the sialic acid-binding site on HA was determined by lack of a red dot in the well, which indicates formation of a lattice-structure comprising RBCs and either virus or HA-ferritin nanoparticles. The results of this analysis, which are shown in FIG. 46, demonstrate that the ability of HA-ferritin nanoparticles of the present invention to agglutinate red blood cells is equivalent to that of inactivated influenza virus.

The importance of the HA protein sialic acid binding domain on the ability of the nanoparticles to agglutinate RBCs was examined by constructing nanoparticles using HA-ferritin fusion proteins in which the sialic acid binding domain was disrupted by a mutation. These nanoparticles were then used in an agglutination assay, as described above. The results of this analysis, which are shown in FIG. 46, demonstrate that the sialic acid binding domain is essential to the ability of the nanoparticles to agglutinate RBCs.

The ability of HA-ferritin nanoparticles to be used in a hemagglutinin inhibition assay was compared to that of live virus as follows:

HAI Assay Using Live Virus

Seed stocks of influenza viruses were obtained from the CDC (Atlanta, Ga.) and the viruses expanded in embryonated chicken eggs or in Madin-Darby canine kidney (MDCK) cells. Serum samples were pretreated with receptor-destroying enzyme (RDE II; Denka Seiken Co., Ltd.) overnight at 37° C. followed by heat inactivation at 56° C. for 30 minutes. HAI assays were conducted in V-bottom 96-well plates (Corning, Inc.) using four hemagglutinating units of virus per well and 0.5% turkey or chicken red blood cells (RBCs). Sera pretreated with RDE were serially diluted in PBS (25 μl) and mixed with virus (25 μl) for 30 min at room temperature. RBCs (50 μl) were then added to the reaction and incubated for another 30 min at room temperature. Hemagglutination was determined by visual inspection of the wells for the presence (inhibition; anti-influenza antibodies present) or absence (lack of inhibition; no anti-influenza antibodies present) of a red dot in the bottom of the well.

HAI Assay Using HA-Ferritin Nanoparticles

HA-ferritin nanoparticles were produced using the methods disclosed herein. Briefly, vectors expressing influenza HA were transfected into 293F cells (Invitrogen) using 293fectin (Invitrogen) according to the manufacturer's instructions. Matched neuraminidase (NA) vectors were co-transfected at a ratio of 20:1 HA:NA (wt:wt) (NA cleaves the sialic acid from the HA to prevent the HA proteins from binding to each other and forming aggregates). The cells were grown in Freestyle 293 expression medium (Invitrogen) and the culture supernatants were collected 4 days post-transfection. The supernatants were concentrated and then buffer exchanged to PBS. The HA ferritin-nanoparticles were purified by affinity column chromatography using Erythrina cristagalli agglutinin (ECA, coral tree lectin; EY Laboratories, Inc.) specific for galactose β(1,4) N-acetylglucosamine. The HA ferritin-nanoparticles were further purified by size exclusion chromatography with a Superose 6 PG XK 16/70 column (GE Healthcare) in PBS. HAI assays were performed as described above, using V-bottom 96-well plates (Corning, Inc.) and four hemagglutinating units of HA ferritin-nanoparticles per well.

The HA-ferritin nanoparticles were then tested for their ability to work in a HAI assay. Briefly, serum samples were pretreated with receptor-destroying enzyme (RDE II; Denka Seiken Co., Ltd.) overnight at 37° C. followed by heat inactivation at 56° C. for 30 minutes. HAI assays were conducted in V-bottom 96-well plates (Corning, Inc.) using four hemagglutinating units of HA ferritin nanoparticles per well and 0.5% turkey or chicken red blood cells (RBCs). Sera pretreated with RDE were serially diluted in PBS (25 μl) and mixed with HA ferritin nanoparticles (25 μl) for 30 min at room temperature. RBCs (50 μl) were then added to the reaction and incubated for another 30 min at room temperature. Hemagglutination was determined by visual inspection of the wells for the presence (inhibition; anti-influenza antibodies present) or absence (lack of inhibition; no anti-influenza antibodies present) of a red dot in the bottom of the well.

The results of these studies, which are shown in FIG. 48, demonstrate that the level of anti-influenza antibodies obtained using the HA-ferritin nanoparticle HAI assay is highly correlated with the level obtained using a conventional virus HAI assay. 

What is claimed:
 1. A method for detecting anti-influenza virus antibodies in a sample, the method comprising: a. contacting at least a portion of the sample with a nanoparticle comprising a fusion protein, wherein the fusion protein comprises at least 25 contiguous amino acids from a monomeric ferritin subunit protein joined to at least one epitope from an influenza hemagglutinin (HA) protein such that the nanoparticle comprises trimers of the influenza virus HA protein epitope on its surface, and wherein the at least a portion of the sample and the nanoparticle are contacted under conditions suitable for forming a complex between the nanoparticle and the anti-influenza virus antibodies, if present; and, b. analyzing the contacted sample for the presence of a nanoparticle/antibody complex, wherein the presence of such a complex indicates the sample contains anti-influenza antibodies.
 2. The method of claim 1, wherein the monomeric ferritin subunit protein is selected from the group consisting of a bacterial ferritin, a plant ferritin, an algal ferritin and a mammalian ferritin.
 3. The nanoparticle of claim 1, wherein the monomeric ferritin subunit protein comprises at least 25 contiguous amino acids an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5, where in the fusion protein is capable of self-assembling into nanoparticles.
 4. The nanoparticle of claim 1, wherein the hemagglutinin protein is from an influenza virus selected from the group consisting of A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009 CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968 (1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo, B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris, H1), B/Brisbane/60/2008 (2008 Bris, B).
 5. The nanoparticle of claim 1, wherein the fusion protein comprises an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, wherein the nanoparticle elicits an immune response against an influenza virus.
 6. The nanoparticle of claim 1, wherein the nanoparticle comprises a second fusion protein comprising a second influenza hemagglutinin protein, wherein the first and second influenza hemagglutinin proteins are from different types, sub-types or strains of influenza viruses.
 7. The method of claim 1, wherein the step of analyzing comprises an assay selected from the group consisting of a hemagglutinin inhibition assay, an immunodiffusion assay, an enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, a precipitation assay, an immunoprecipitation assay, a BioCoreJ assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG=s Immunodot System, Fribourg, Switzerland), an immunoblot assay (e.g., a western blot), an phosphorescence assay, a chromatography assay, a PAGe-based assay, a surface plasmon resonance assay, a spectrophotometric assay, and an electronic sensory assay.
 8. A method for identifying an individual having anti-influenza antibodies, comprising: a. contacting at least a portion of a sample from an individual with a nanoparticle comprising a fusion protein, wherein the fusion protein comprises at least 25 contiguous amino acids from a monomeric ferritin subunit protein joined to at least one epitope from an influenza hemagglutinin (HA) protein such that the nanoparticle comprises trimers of the influenza virus HA protein epitope on its surface, and wherein the at least a portion of the sample and the nanoparticle are contacted under conditions suitable for forming a complex between the nanoparticle and the anti-influenza virus antibodies, if present; b. analyzing the contacted sample for the presence of a nanoparticle/antibody complex, wherein the presence of such a complex indicates the individual has anti-influenza antibodies.
 9. The method of claim 8, wherein the monomeric ferritin subunit protein is selected from the group consisting of a bacterial ferritin, a plant ferritin, an algal ferritin and a mammalian ferritin.
 10. The nanoparticle of claim 8, wherein the monomeric ferritin subunit protein comprises at least 25 contiguous amino acids an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5, where in the fusion protein is capable of self-assembling into nanoparticles.
 11. The nanoparticle of claim 8, wherein the hemagglutinin protein is from an influenza virus selected from the group consisting of A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009 CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968 (1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo, B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris, H1), B/Brisbane/60/2008 (2008 Bris, B).
 12. The nanoparticle of claim 8, wherein the fusion protein comprises an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, wherein the nanoparticle elicits an immune response against an influenza virus.
 13. The nanoparticle of claim 8, wherein the nanoparticle comprises a second fusion protein comprising a second influenza hemagglutinin protein, wherein the first and second influenza hemagglutinin proteins are from different types, sub-types or strains of influenza viruses.
 14. The method of claim 8, wherein the step of analyzing comprises an assay selected from the group consisting of a hemagglutinin inhibition assay, an immunodiffusion assay, an enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, a precipitation assay, an immunoprecipitation assay, a BioCoreJ assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG=s Immunodot System, Fribourg, Switzerland), an immunoblot assay (e.g., a western blot), an phosphorescence assay, a chromatography assay, a PAGe-based assay, a surface plasmon resonance assay, a spectrophotometric assay, and an electronic sensory assay.
 15. A method for measuring the response of an individual to a vaccine, comprising: a. administering to the individual a vaccine for influenza virus; b. contacting at least a portion of a sample from the individual with a nanoparticle comprising a fusion protein, wherein the fusion protein comprises at least 25 contiguous amino acids from a monomeric ferritin subunit protein joined to at least one epitope from an influenza hemagglutinin (HA) protein such that the nanoparticle comprises trimers of the influenza virus HA protein epitope on its surface, and wherein the at least a portion of the sample and the nanoparticle are contacted under conditions suitable for forming a complex between the nanoparticle and the anti-influenza virus antibodies, if present; and, c. determining the level of antibody present in the sample by determining the level of nanoparticle/antibody complex; wherein an increase in the level of antibody in the sample over the pre-vaccination level of antibody indicates the vaccine induced an immune response in the individual.
 16. The method of claim 15, wherein the monomeric ferritin subunit protein is selected from the group consisting of a bacterial ferritin, a plant ferritin, an algal ferritin and a mammalian ferritin.
 17. The nanoparticle of claim 15, wherein the monomeric ferritin subunit protein comprises at least 25 contiguous amino acids an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5, where in the fusion protein is capable of self-assembling into nanoparticles.
 18. The nanoparticle of claim 15, wherein the hemagglutinin protein is from an influenza virus selected from the group consisting of A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009 CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968 (1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo, B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris, H1), B/Brisbane/60/2008 (2008 Bris, B).
 19. The nanoparticle of claim 15, wherein the fusion protein comprises an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, wherein the nanoparticle elicits an immune response against an influenza virus.
 20. The method of claim 15, wherein the step of analyzing comprises an assay selected from the group consisting of a hemagglutinin inhibition assay, an immunodiffusion assay, an enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, a precipitation assay, an immunoprecipitation assay, a BioCoreJ assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG=s Immunodot System, Fribourg, Switzerland), an immunoblot assay (e.g., a western blot), an phosphorescence assay, a chromatography assay, a PAGe-based assay, a surface plasmon resonance assay, a spectrophotometric assay, and an electronic sensory assay. 